Speed of lung inflation at birth influences the initiation of lung injury in preterm lambs.

Gas flow is fundamental for driving tidal ventilation and, thus, the speed of lung motion, but current bias flow settings to support the preterm lung after birth do not have an evidence base. We aimed to determine the role of gas bias flow rates to generate positive pressure ventilation in initiating early lung injury pathways in the preterm lamb. Using slower speeds to inflate the lung during tidal ventilation (gas flow rates 4-6 L/min) did not affect lung mechanics, mechanical power, or gas exchange compared with those currently used in clinical practice (8-10 L/min). Speed of pressure and volume change during inflation were faster with higher flow rates. Lower flow rates resulted in less bronchoalveolar fluid protein, better lung morphology, and fewer detached epithelial cells. Overall, relative to unventilated fetal controls, there was greater protein change using 8-10 L/min, which was associated with enrichment of acute inflammatory and innate responses. Slowing the speed of lung motion by supporting the preterm lung from birth with lower flow rates than in current clinical use resulted in less lung injury without compromising tidal ventilation or gas exchange.

Plasma Proteome Profiles Associated with Early Development of Lung Injury in Extremely Preterm Infants.

The biological mediators which initiate lung injury in extremely preterm infants during early postnatal life remain largely unidentified, limiting opportunities for early treatment and diagnosis. This exploratory study used SWATH-mass spectrometry to identify bronchopulmonary dysplasia (BPD)-specific changes in protein abundance in plasma samples obtained in the first 72 hours of life from extremely preterm infants and bioinformatic analysis to identify BPD-related biological categories and pathways. Lasty, binary logistic regression analysis was used to test the BPD predictive potential of a base model alone (gestational age, birth weight, sex) and with the protein biomarker added, with bootstrap resampling used to internally validate protein predictors and adjust for overoptimism. We observed disturbance of key processes including coagulation, complement activation, development and extracellular matrix organisation in the first days of life in extremely preterm infants who were later diagnosed with BPD. In the BPD prediction analysis, 49 plasma proteins were identified which when each singularly was combined with birth characteristics had a C-index of 0.65-0.84 (optimism-adjusted C-index) suggesting predictive potential for BPD outcomes. Taken together, this study demonstrates that alterations in plasma proteins can be detected from 4 hours of age in extremely preterm infants who later develop BPD and that protein biomarkers when combined with three birth characteristics have the potential to predict BPD development within the first 72 hours of life.

Lung ultrasound detects regional aeration inhomogeneity in ventilated preterm lambs.

BACKGROUND: Inhomogeneous lung aeration is a significant contributor to preterm lung injury. EIT detects inhomogeneous aeration in the research setting. Whether LUS detects inhomogeneous aeration is unknown. The aim was to determine whether LUS detects regional inhomogeneity identified by EIT in preterm lambs. METHODS: LUS and EIT were simultaneously performed on mechanically ventilated preterm lambs. LUS images from non-dependent and dependent regions were acquired and reported using a validated scoring system and computer-assisted quantitative LUS greyscale analysis (Q-LUSMGV). Regional inhomogeneity was calculated by observed over predicted aeration ratio from the EIT reconstructive model. LUS scores and Q-LUSMGV were compared with EIT aeration ratios using one-way ANOVA. RESULTS: LUS was performed in 32 lambs (~125d gestation, 128 images). LUS scores were greater in upper anterior (non-dependent) compared to lower lateral (dependent) regions of the left (3.4 vs 2.9, p = 0.1) and right (3.4 vs 2.7, p < 0.0087). The left and right upper regions also had greater LUS scores compared to right lower (3.4 vs 2.7, p < 0.0087) and left lower (3.7 vs 2.9, p = 0.1). Q-LUSMGV yielded similar results. All LUS findings corresponded with EIT regional differences. CONCLUSION: LUS may have potential in measuring regional aeration, which should be further explored in human studies. IMPACT: Inhomogeneous lung aeration is an important contributor to preterm lung injury, however, tools detecting inhomogeneous aeration at the bedside are limited. Currently, the only tool clinically available to detect this is electrical impedance tomography (EIT), however, its use is largely limited to research. Lung ultrasound (LUS) may play a role in monitoring lung aeration in preterm infants, however, whether it detects inhomogeneous lung aeration is unknown. Visual LUS scores and mean greyscale image analysis using computer assisted quantitative LUS (Q-LUSMGV) detects regional lung aeration differences when compared to EIT. This suggests LUS reliably detects aeration inhomogeneity warranting further investigation in human trials.

Inflating Pressure and Not Expiratory Pressure Initiates Lung Injury at Birth in Preterm Lambs.

Rationale: Inflation is essential for aeration at birth, but current inflating pressure settings are without an evidence base. Objectives: To determine the role of inflating pressure (ΔP), and its relationship with positive end-expiratory pressure (PEEP), in initiating early lung injury pathways in the preterm lamb lung. Methods: Preterm (124 to 127 d) steroid-exposed lambs (n = 45) were randomly allocated (8-10 per group) to 15 minutes of respiratory support with placental circulation and 20 or 30 cm H2O ΔP, with an initial high PEEP (maximum, 20 cm H2O) recruitment maneuver known to facilitate aeration (dynamic PEEP), and compared with dynamic PEEP with no ΔP or 30 cm H2O ΔP and low (4 cm H2O) PEEP. Lung mechanics and aeration were measured throughout. After an additional 30 minutes of apneic placental support, lung tissue and bronchoalveolar fluid were analyzed for regional lung injury, including proteomics. Measurements and Main Results: The 30 cm H2O ΔP and dynamic PEEP strategies resulted in quicker aeration and better compliance but higher tidal volumes (often >8 ml/kg, all P < 0.0001; mixed effects) and injury. ΔP 20 cm H2O with dynamic PEEP resulted in the same lung mechanics and aeration, but less energy transmission (tidal mechanical power), as ΔP 30 cm H2O with low PEEP. Dynamic PEEP without any tidal inflations resulted in the least lung injury. Use of any tidal inflating pressures altered metabolic, coagulation and complement protein pathways within the lung. Conclusions: Inflating pressure is essential for the preterm lung at birth, but it is also the primary mediator of lung injury. Greater focus is needed on strategies that identify the safest application of pressure in the delivery room.

Respiratory strategy at birth initiates distinct lung injury phenotypes in the preterm lamb lung.

BACKGROUND: A lack of clear trial evidence often hampers clinical decision-making during support of the preterm lung at birth. Protein biomarkers have been used to define acute lung injury phenotypes and improve patient selection for specific interventions in adult respiratory distress syndrome. The objective of the study was to use proteomics to provide a deeper biological understanding of acute lung injury phenotypes resulting from different aeration strategies at birth in the preterm lung. METHODS: Changes in protein abundance against an unventilated group (n = 7) were identified via mass spectrometry in a biobank of gravity dependent and non-dependent lung tissue from preterm lambs managed with either a Sustained Inflation (SI, n = 20), Dynamic PEEP (DynPEEP, n = 19) or static PEEP (StatPEEP, n = 11). Ventilation strategy-specific pathways and functions were identified (PANTHER and WebGestalt Tool) and phenotypes defined using integrated analysis of proteome, physiological and clinical datasets (MixOmics package). RESULTS: 2372 proteins were identified. More altered proteins were identified in the non-dependent lung, and in SI group than StatPEEP and DynPEEP. Different inflammation, immune system, apoptosis and cytokine pathway enrichment were identified for each strategy and lung region. Specific integration maps of clinical and physiological outcomes to specific proteins could be generated for each strategy. CONCLUSIONS: Proteomics mapped the molecular events initiating acute lung injury and identified detailed strategy-specific phenotypes. This study demonstrates the potential to characterise preterm lung injury by the direct aetiology and response to lung injury; the first step towards true precision medicine in neonatology.

Dynamic positive end-expiratory pressure strategies using time and pressure recruitment at birth reduce early expression of lung injury in preterm lambs.

Positive end-expiratory pressure (PEEP) is critical to the preterm lung at birth, but the optimal PEEP level remains uncertain. The objective of this study was to determine the effect of maximum PEEP levels at birth on the physiological and injury response in preterm lambs. Steroid-exposed preterm lambs (124-127 days gestation; n = 65) were randomly assigned from birth to either 1) positive pressure ventilation (PPV) at 8 cmH2O PEEP or 3-min dynamic stepwise PEEP strategy (DynPEEP), with either 2) 20 cmH2O maximum PEEP (10 PEEP second steps) or 3) 14 cmH2O maximum PEEP (20-s steps), all followed by standardized PPV for 90 min. Lung mechanics, gas exchange, regional ventilation and aeration (electrical impedance tomography), and histological and molecular measures of lung injury were compared between groups. Dynamic compliance was greatest using a maximum 20 cmH2O (DynPEEP). There were no differences in gas exchange, end-expiratory volume, and ventilator requirements. Regional ventilation became more uniform with time following all PEEP strategies. For all groups, gene expression of markers of early lung injury was greater in the gravity nondependent lung, and inversely related to the magnitude of PEEP, being lowest in the 20 cmH2O DynPEEP group overall. PEEP levels had no impact on lung injury in the dependent lung. Transient high maximum PEEP levels using dynamic PEEP strategies may confer more lung protection at birth.

Regional ventilation characteristics during non-invasive respiratory support in preterm infants.

OBJECTIVES: To determine the regional ventilation characteristics during non-invasive ventilation (NIV) in stable preterm infants. The secondary aim was to explore the relationship between indicators of ventilation homogeneity and other clinical measures of respiratory status. DESIGN: Prospective observational study. SETTING: Two tertiary neonatal intensive care units. PATIENTS: Forty stable preterm infants born <30 weeks of gestation receiving either continuous positive airway pressure (n=32) or high-flow nasal cannulae (n=8) at least 24 hours after extubation at time of study. INTERVENTIONS: Continuous electrical impedance tomography imaging of regional ventilation during 60 min of quiet breathing on clinician-determined non-invasive settings. MAIN OUTCOME MEASURES: Gravity-dependent and right-left centre of ventilation (CoV), percentage of whole lung tidal volume (VT) by lung region and percentage of lung unventilated were determined for 120 artefact-free breaths/infant (4770 breaths included). Oxygen saturation, heart and respiratory rates were also measured. RESULTS: Ventilation was greater in the right lung (mean 69.1 (SD 14.9)%) total VT and the gravity-non-dependent (ND) lung; ideal-actual CoV 1.4 (4.5)%. The central third of the lung received the most VT, followed by the non-dependent and dependent regions (p<0.0001 repeated-measure analysis of variance). Ventilation inhomogeneity was associated with worse peripheral capillary oxygen saturation (SpO2)/fraction of inspired oxygen (FiO2) (p=0.031, r2 0.12; linear regression). In those infants that later developed bronchopulmonary dysplasia (n=25), SpO2/FiO2 was worse and non-dependent ventilation inhomogeneity was greater than in those that did not (both p<0.05, t-test Welch correction). CONCLUSIONS: There is high breath-by-breath variability in regional ventilation patterns during NIV in preterm infants. Ventilation favoured the ND lung, with ventilation inhomogeneity associated with worse oxygenation.

Proteomics reveals region-specific hemostatic alterations in response to mechanical ventilation in a preterm lamb model of lung injury.

INTRODUCTION: Preterm infants often require assisted ventilation, however ventilation when applied to the immature lung can initiate ventilator-induced lung injury (VILI). The biotrauma which underscores VILI is largely undefined, and is likely to involve vascular injury responses, including hemostasis. We aimed to use a ventilated, preterm lamb model to: (1) characterize regional alterations in hemostatic mediators within the lung and (2) assess the functional impact of protein alterations on hemostasis by analyzing temporal thrombin generation. MATERIALS AND METHODS: Preterm lambs delivered at 124 to 127 days gestation received 90 min of mechanical ventilation (positive end-expiratory pressure = 8 cm H2O, VT = 6-8 ml/kg) and were compared with unventilated control lambs. At study completion, lung tissue was taken from standardized nondependent and gravity-dependent regions, and Orbitrap-mass spectrometry and KEGG were used to identify and map regional alterations in hemostasis pathway members. Temporal alterations in plasma thrombin generation were assessed. RESULTS: Ventilation was distributed towards the nondependent lung. Significant changes in hemostatic protein abundance, were detected at a two-fold higher rate in the nondependent lung when compared with the gravity-dependent lung. Seven proteins were uniquely altered in non-dependent lung (SERPINA1, MYL12A, RAP1B, RHOA, ITGB1, A2M, GNAI2), compared with a single proteins in gravity-dependent lung (COL1A2). Four proteins were altered in both regions (VTN, FGG, FGA, and ACTB). Tissue protein alterations were mirrored by plasma hypocoagulability at 90-minutes of ventilation. CONCLUSIONS: We observed regionally specific, hemostatic alterations within the preterm lung together with disturbed fibrinolysis following a short period of mechanical ventilation.

Respiratory mechanics during initial lung aeration at birth in the preterm lamb.

Despite recent insights into the dynamic processes during lung aeration at birth, several aspects remain poorly understood. We aimed to characterize changes in lung mechanics during the first inflation at birth and their relationship to changes in lung volume. Intubated preterm lambs (gestational age, 124-127 days; n = 17) were studied at birth. Lung volume changes were measured by electrical impedance tomography (VLEIT). Respiratory system resistance (R5) and oscillatory compliance (Cx5) were monitored with the forced oscillation technique at 5 Hz. Lambs received 3-7 s of 8 cmH2O of continuous distending pressure (CDP) before delivery of a sustained inflation (SI) of 40 cmH2O. The SI was then applied until either Cx5 or the VLEIT or the airway opening volume was stable. CDP was resumed for 3-7 s before commencement of mechanical ventilation. The exponential increases with time of Cx5 and VLEIT from commencement of the SI were characterized by estimating their time constants (τCx5 and τVLEIT, respectively). During SI, a fast decrease in R5 and an exponential increase in Cx5 and VLEIT were observed. Cx5 and VLEIT provided comparable information on the dynamics of lung aeration in all lambs, with τCx5 and τVLEIT being highly linearly correlated (r2 = 0.87, P < 0.001). Cx5 and VLEIT decreased immediately after SI. Despite the standardization of the animal model, changes in Cx5 and R5 both during and after SI were highly variable. Lung aeration at birth is characterized by a fast reduction in resistance and a slower increase in oscillatory compliance, the latter being a direct reflection of the amount of lung aeration.

A dedicated respiratory function monitor to improve tidal volume delivery during neonatal anesthesia.

BACKGROUND: Tight control of tidal volume using accurate monitoring may improve neonatal outcomes. However, respiratory function monitors incorporated in current anesthetic workstations are generally inaccurate at tidal volumes used for infants. AIMS: To determine if a specific respiratory function monitor for neonatal infants improved expired tidal volume delivery during anesthesia. METHOD: Infants <3 months old requiring intubation for surgery in the operating theater were studied. After intubation a Phillips NM3, Acutronic Florian, or Novametrix Ventcheck Respiratory Function Monitor was integrated into the circuit, and clinicians given access to the display for the duration of anesthesia. Breath-to-breath expired tidal volume delivery, leak, and delivered pressure were recorded, with cardiorespiratory parameters. These were compared with a matched control group with clinicians blinded to respiratory function monitor display. RESULTS: A total of 10 055 and 2569 inflations were measured in the respiratory function monitor visible (n = 32) and masked (n = 33) groups, respectively, with mean (standard deviation) delivered expired tidal volume 7.5 (2.4) mL/kg and 7.7 (3.0) mL/kg, respectively; mean difference (95% confidence interval) -0.2 (-1.1, 0.8) mL/kg (Welch's t test). In the visible group, 55.6% of expired tidal volumes were between 4 and 8 mL/kg compared to 51.7% in the masked group; relative benefit (95% confidence interval), 1.08 (1.03, 1.12). Expired tidal volume was less likely to be <4 mL/kg in the visible group compared to masked group; 6.4% vs 9.8%, 1.53 (1.33, 1.76). The use of a respiratory function monitor also reduced the number of inflations >10 mL/kg; 13.0% vs 22.0%, 1.11 (1.09, 1.14). CONCLUSION: Tidal volumes <4 mL/kg and >10 mL/kg are frequently delivered during neonatal anesthesia. The inclusion of an accurate respiratory function monitor may reduce the risk of exposure to potentially harmful tidal volumes.

Preterm Lung Exhibits Distinct Spatiotemporal Proteome Expression at Initiation of Lung Injury.

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 H2O, 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.

Gradual Aeration at Birth Is More Lung Protective Than a Sustained Inflation in Preterm Lambs.

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 ± 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 H2O. 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 No-RM, 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 No-RM 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.

Surgery and magnetic resonance imaging increase the risk of hypothermia in infants.

AIM: Maintaining normothermia is a tenet of neonatal care. However, neonatal thermal care guidelines applicable to intra-hospital transport beyond the neonatal intensive care unit (NICU) and during surgery or magnetic resonance imaging (MRI) are lacking. The aim of this study is to determine the proportion of infants normothermic (36.5-37.5°C) on return to NICU after management during surgery and MRI, and during standard clinical care in both environments. METHODS: Sixty-two newborns requiring either surgery in the operating theatre (OT) (n = 41) or an MRI scan (n = 21) at the Royal Children's Hospital (Melbourne) NICU were prospectively studied. Core temperature, along with cardiorespiratory parameters, was continuously measured from 15 min prior to leaving the NICU until 60 min after returning. Passive and active warming (intra-operatively) was at clinician discretion. RESULTS: The study reported 90% of infants were normothermic before leaving NICU: 86% (MRI) and 93% (OT). Only 52% of infants were normothermic on return to NICU (relative risk (RR) 1.75; 95% confidence interval (CI) 1.39-2.31; number needed to harm (NNH) 2.6). Between departure from the NICU and commencement of surgery, core temperature decreased by mean 0.81°C (95% CI 0.30-1.33; P = 0.0001, analysis of variance), with only 24% of infants normothermic when surgery began (P < 0.0001; RR 3.80 (95% CI 2.33-6.74); NNH 1.5). After an MRI, infants were a mean 0.41°C (95% CI 0.16-0.67) colder than immediately before entering the scanner (P = 0.001, analysis of variance), with only 43% being normothermic (P = 0.003; RR 2.11 (95% CI 1.35-3.74); NNH 2.1). CONCLUSION: Unintentional hypothermia is a common occurrence during surgery in the OT and MRI in neonates, indicating that evidence-based warming strategies to prevent hypothermia should be developed.

Farnesoid X Receptor Agonist Treatment Alters Bile Acid Metabolism but Exacerbates Liver Damage in a Piglet Model of Short-Bowel Syndrome.

BACKGROUND & AIMS: Options for the prevention of short-bowel syndrome-associated liver disease (SBS-ALDs) are limited and often ineffective. The farnesoid X receptor (FXR) is a newly emerging pharmaceutical target and FXR agonists have been shown to ameliorate cholestasis and metabolic disorders. The aim of this study was to assess the efficacy of obeticholic acid (OCA) treatment in preventing SBS-ALDs. METHODS: Piglets underwent 75% small-bowel resection (SBS) or sham surgery (sham) and were assigned to either a daily dose of OCA (2.4 mg/kg/day) or were untreated. Clinical measures included weight gain and stool studies. Histologic features were assessed. Ultraperformance liquid chromatography tandem mass spectrometry was used to determine bile acid composition in end point bile and portal serum samples. Gene expression of key FXR targets was assessed in intestinal and hepatic tissues via quantitative polymerase chain reaction. RESULTS: OCA-treated SBS piglets showed decreased stool fat and altered liver histology when compared with nontreated SBS piglets. OCA prevented SBS-associated taurine depletion, however, further analysis of bile and portal serum samples indicated that OCA did not prevent SBS-associated alterations in bile acid composition. The expression of FXR target genes involved in bile acid transport and synthesis increased within the liver of SBS piglets after OCA administration whereas, paradoxically, intestinal expression of FXR target genes were decreased by OCA administration. CONCLUSIONS: Administration of OCA in SBS reduced fat malabsorption and altered bile acid composition, but did not prevent the development of SBS-ALDs. We postulate that extensive small resection impacts the ability of the remnant intestine to respond to FXR activation.

Tidal Volume Delivery during the Anesthetic Management of Neonates Is Variable.

OBJECTIVES: To describe expiratory tidal volume (VT) during routine anesthetic management of neonates at a single tertiary neonatal surgical center, as well as the proportion of VT values within the range of 4.0-8.0 mL/kg. STUDY DESIGN: A total of 26 neonates needing surgery under general anesthesia were studied, of whom 18 were intubated postoperatively. VT was measured continuously during normal clinical care using a dedicated neonatal respiratory function monitor (RFM), with clinicians blinded to values. VT, pressure, and cardiorespiratory variables were recorded regularly while intubated intraoperatively, during postoperative transport, and for 15 minutes after returning to the neonatal intensive care unit (NICU). In addition, paired VT values from the anesthetic machine were documented intraoperatively. RESULTS: A total of 2597 VT measures were recorded from 26 neonates. Intraoperative and postoperative transport expiratory VT values were highly variable compared with the NICU VT (P < .0001, Kruskal-Wallis test), with 51% of inflations outside the 4.0-8.0 mL/kg range (35% and 38% of VT >8.0 mL/kg, respectively), compared with 29% in the NICU (P < .001, χ2 test). The use of a flow-inflating bag resulted in a median (range) VT of 8.5 mL/kg (range, 5.3-11.4 mL/kg) vs 5.6 ml/kg (range, 4.3-7.9 mL/kg) using a Neopuff T-piece system (P < .0001, Mann-Whitney U test). The mean anesthetic machine expiratory VT was 3.2 mL/kg (95% CI, -4.5 to 10.8 mL/kg) above RFM. CONCLUSIONS: VT is highly variable during the anesthetic care of neonates, and potentially injurious VT is frequently delivered; thus, we suggest close VT monitoring using a dedicated neonatal RFM.

Effectiveness of individualized lung recruitment strategies at birth: an experimental study in preterm lambs.

Respiratory transition at birth involves rapidly clearing fetal lung liquid and preventing efflux back into the lung while aeration is established. We have developed a sustained inflation (SIOPT) individualized to volume response and a dynamic tidal positive end-expiratory pressure (PEEP) (open lung volume, OLV) strategy that both enhance this process. We aimed to compare the effect of each with a group managed with PEEP of 8 cmH2O and no recruitment maneuver (No-RM), on gas exchange, lung mechanics, spatiotemporal aeration, and lung injury in 127 ± 1 day preterm lambs. Forty-eight fetal-instrumented lambs exposed to antenatal steroids were ventilated for 60 min after application of the allocated strategy. Spatiotemporal aeration and lung mechanics were measured with electrical impedance tomography and forced-oscillation, respectively. At study completion, molecular and histological markers of lung injury were analyzed. Mean (SD) aeration at the end of the SIOPT and OLV groups was 32 (22) and 38 (15) ml/kg, compared with 17 (10) ml/kg (180 s) in the No-RM (P = 0.024, 1-way ANOVA). This translated into better oxygenation at 60 min (P = 0.047; 2-way ANOVA) resulting from better distal lung tissue aeration in SIOPT and OLV. There was no difference in lung injury. Neither SIOPT nor OLV achieved homogeneous aeration. Histological injury and mRNA biomarker upregulation were more likely in the regions with better initial aeration, suggesting volutrauma. Tidal ventilation or an SI achieves similar aeration if optimized, suggesting that preventing fluid efflux after lung liquid clearance is at least as important as fluid clearance during the initial inflation at birth.

Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model.

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 proteomics of lung injury in childhood: challenges and opportunities.

Proteomics, the large-scale study of the structure and function of proteins of a cell or organism, is a rapidly developing area of biomedical research which is perfectly suited to the study of pediatric lung injury, where a variety of samples are easily, and repeatedly, accessible including plasma (reflecting a whole body response) and broncheoalveolar lung fluid (reflecting the lungs response). When applied to pediatric lung injury, proteomics could be used to develop much needed early biomarkers of lung injury, elucidate pathological pathways and determine protein alterations associated with specific disease processes. However despite the obvious benefits and need, proteomics is rarely utilized in studies of pediatric injury. This review primarily reports on the last decade of pediatric research into proteomes associated with specific respiratory diseases including bronchopulmonary dysplasia, respiratory infection, cystic fibrosis and asthma whilst also reflecting on the challenges unique to proteomic studies of the pediatric respiratory disease population. We conclude that the number of key pathological differences between the pediatric and adult study populations inhibit inference of results from adult studies onto a pediatric population and necessitate studies of the pediatric proteome. Furthermore the disparity amongst pediatric lung disease in terms of age at onset and underlying pathological mechanism (genetic, immunological, intervention-based, developmental arrest, inhaled toxin) will require proteomic studies which are well designed, with large disease specific patient sets to ensure adequate power as well as matched controls. Regardless of causative agent, pulmonary biomarkers are needed to predict the clinical course of pediatric lung disease, status, progression and response to treatment. Identification of early biomarkers is particularly pertinent in order to understand the natural history of disease and monitor progression so prevention of ongoing lung injury and impact on childhood can targeted.

Changes in the colon microbiota and intestinal cytokine gene expression following minimal intestinal surgery.

AIM: To investigate the impact of minor abdominal surgery on the caecal microbial population and on markers of gut inflammation. METHODS: Four week old piglets were randomly allocated to a no-surgery "control" group (n = 6) or a "transection surgery" group (n = 5). During the transection surgery procedure, a conventional midline incision of the lower abdominal wall was made and the small intestine was transected at a site 225 cm proximal to the ileocaecal valve, a 2 cm segment was removed and the intestine was re-anastomosed. Piglets received a polymeric infant formula diet throughout the study period and were sacrificed at two weeks post-surgery. Clinical outcomes including weight, stool consistency and presence of stool fat globules were monitored. High throughput DNA sequencing of colonic content was used to detect surgery-related disturbances in microbial composition at phylum, family and genus level. Diversity and richness estimates were calculated for the control and minor surgery groups. As disturbances in the gut microbial community are linked to inflammation we compared the gene expression of key inflammatory cytokines (TNF, IL1B, IL18, IL12, IL8, IL6 and IL10) in ileum, terminal ileum and colon mucosal extracts obtained from control and abdominal surgery groups at two weeks post-surgery. RESULTS: Changes in the relative abundance of bacterial species at family and genus level were confined to bacterial members of the Proteobacteria and Bacteroidetes phyla. Family level compositional shifts included a reduction in the relative abundance of Enterobacteriaceae (22.95 ± 5.27 vs 2.07 ± 0.72, P < 0.01), Bacteroidaceae (2.54 ± 0.56 vs 0.86 ± 0.43, P < 0.05) and Rhodospirillaceae (0.40 ± 0.14 vs 0.00 ± 0.00, P < 0.05) following transection surgery. Similarly, at the genus level, changes associated with transection surgery were restricted to members of the Proteobacteria and Bacteroidetes phyla and included decreased relative abundance of Enterobacteriaceae (29.20 ± 6.74 vs 2.88 ± 1.08, P < 0.01), Alistipes (4.82 ± 1.73 vs 0.18 ± 0.13, P < 0.05) and Thalassospira (0.53 ± 0.19 vs 0.00 ± 0.00, P < 0.05). Surgery-associated microbial dysbiosis was accompanied by increased gene expression of markers of inflammation. Within the ileum IL6 expression was decreased (4.46 ± 1.60 vs 0.24 ± 0.06, P < 0.05) following transection surgery. In the terminal ileum, gene expression of TNF was decreased (1.51 ± 0.13 vs 0.80 ± 0.16, P < 0.01) and IL18 (1.21 ± 0.18 vs 2.13 ± 0.24, P < 0.01), IL12 (1.04 ± 0.16 vs 1.82 ± 0.32, P < 0.05) and IL10 (1.04 ± 0.06 vs 1.43 ± 0.09, P < 0.01) gene expression increased following transection surgery. Within the colon, IL12 (0.72 ± 0.13 vs 1.78 ± 0.28, P < 0.01) and IL10 (0.98 ± 0.02 vs 1.95 ± 0.14, P < 0.01) gene expression were increased following transection surgery. CONCLUSION: This study suggests that minor abdominal surgery in infants, results in long-term alteration of the colonic microbial composition and persistent gastrointestinal inflammation.

Gut microbial diversity is reduced and is associated with colonic inflammation in a piglet model of short bowel syndrome.

BACKGROUND AND OBJECTIVES: Following small bowel resection (SBR), the luminal environment is altered, which contributes to clinical manifestations of short bowel syndrome (SBS) including malabsorption, mucosal inflammation and bacterial overgrowth. However, the impact of SBR on the colon has not been well-defined. The aims of this study were to characterize the colonic microbiota following SBR and to assess the impact of SBR on mucosal inflammation in the colon. RESULTS: Analysis of the colonic microbiota demonstrated that there was a significant level of dysbiosis both two and six weeks post-SBR, particularly in the phylum Firmicutes, coupled with a decrease in overall bacterial diversity in the colon. This decrease in diversity was associated with an increase in colonic inflammation six weeks post-surgery. METHODS: Female (4-week old) piglets (5-6/group) received a 75% SBR, a transection (sham) or no surgery. Compositional analysis of the colonic microbiota was performed by high-throughput sequencing, two- and six-weeks post-surgery. The gene expression of the pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-8, IL-18 and tumor necrosis factor (TNF)-α in the colonic mucosa was assessed by qRT-PCR and the number of macrophages and percentage inducible nitric oxide synthase (iNOS) staining in the colonic epithelium were quantified by immunohistochemistry. CONCLUSIONS: SBR significantly decreased the diversity of the colonic microbiota and this was associated with an increase in colonic mucosal inflammation. This study supports the hypothesis that SBR has a significant impact on the colon and that this may play an important role in defining clinical outcome.