Association of nursing overtime, nurse staffing and unit occupancy with medical incidents and outcomes of very preterm infants.

OBJECTIVE: To examine the association of nursing overtime, nursing provision and unit occupancy rate with medical incident rates in the neonatal intensive care unit (NICU) and the risk of mortality or major morbidity among very preterm infants. STUDY DESIGN: Single center retrospective cohort study of infants born within 23 to 29 weeks of gestational age or birth weight <1000 g admitted at a 56 bed, level III NICU. Nursing overtime ratios (nursing overtime hours/total nursing hours), nursing provision ratios (nursing hours/recommended nursing hours based on patient dependency categories) and unit occupancy rates were pooled for all shifts during NICU hospitalization of each infant. Log-binomial models assessed their association with the composite outcome (mortality or major morbidity). RESULTS: Of the 257 infants that met the inclusion criteria, 131 (51%) developed the composite outcome. In the adjusted multivariable analyses, high (>3.4%) relative to low nursing overtime ratios (⩽3.4%) were not associated with the composite outcome (relative risk (RR): 0.93; 95% confidence interval (CI): 0.86 to 1.02). High nursing provision ratios (>1) were associated with a lower risk of the composite outcome relative to low ones (⩽1) (RR: 0.81; 95% CI: 0.74 to 0.90). NICU occupancy rates were not associated with the composite outcome (RR: 0.98; 95% CI: 0.89 to 1.07, high (>100%) vs low (⩽100%)). Days with high nursing provision ratios (>1) were also associated with lower risk of having medical incidents (RR: 0.91; 95% CI: 0.82 to 0.99). CONCLUSION: High nursing provision ratio during NICU hospitalization is associated with a lower risk of a composite adverse outcome in very preterm infants.

Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome.

The serial analysis of gene expression (SAGE) method is used to study global gene expression in cells or tissues in various experimental conditions. However, its reproducibility has not yet been definitively assessed. In this study, we have evaluated the reproducibility of the SAGE method and identified the factors that affect it. The determination coefficient (R2 ) for the reproducibility of SAGE is 0.96. However, there are some factors that can affect the reproducibility of SAGE, such as the replication of concatemers and ditags, the number of sequenced tags and double PCR amplification of ditags. Thus, corrections for these factors must be made to ensure the reproducibility and accuracy of SAGE results. A bioinformatic analysis of SAGE data is also presented in order to eliminate these artifacts. Finally, the current study shows that increasing the number of sequenced tags improves the power of the method to detect transcripts and their regulation by experimental conditions.

Association between birth route and late-onset sepsis in very preterm neonates.

OBJECTIVE: To evaluate the association between birth route and late-onset sepsis (LOS), and coagulase-negative Staphylococcal (CONS)-related LOS in preterm neonates. STUDY DESIGN: In this observational study, data from 20,038 infants born between 22 and 32 weeks' gestation and admitted to Canadian neonatal intensive care units between 2010 and 2014 were analyzed retrospectively. The impact of birth route on LOS was assessed using univariate analysis and multiple logistic regression. RESULTS: A total of 8218 neonates were born via vaginal route and 11,820 via cesarean section. Incidence rates of LOS for infants born vaginally and via a cesarean section were 13.1 and 13.2%, respectively, and there was no significant difference in odds of LOS between the groups (adjusted odds ratio (AOR): 0.99; 95% CI 0.87 to 1.12); however, the odds of CONS sepsis were higher in the cesarean group (AOR: 1.15; 95% CI: 1.01 to 1.32). CONCLUSION: Birth route did not have an impact on LOS, but was associated with CONS-related LOS.

Unscheduled apoptosis during acute inflammatory lung injury.

Apoptosis is a mode of cell death currently thought to occur in the absence of inflammation. In contrast, inflammation follows unscheduled events such as acute tissue injury which results in necrosis, not apoptosis. We examined the relevance of this paradigm in three distinct models of acute lung injury; hyperoxia, oleic acid, and bacterial pneumonia. In every case, it was found that apoptosis is actually a prominent component of the acute and inflammatory phase of injury. Moreover, using strains of mice that are differentially sensitive to hyperoxic lung injury we observed that the percent of apoptotic cells was well correlated with the severity of lung injury. These observations suggest that apoptosis may be one of the biological consequences during acute injury and the failure to remove these apoptotic cells may also contribute to the inflammatory response.

P-selectin is upregulated early in the course of hyperoxic lung injury in mice.

While treatment with supplemental oxygen is often essential in patients with lung disease, prolonged therapy may cause lung injury by itself. Although the mechanisms responsible for initiating hyperoxic lung damage almost certainly involve primary oxidative transformations, the possible contributions of inflammation to the tissue injury have been attracting increasing research activity. Increases in intercellular adhesion molecule-1 (ICAM-1) coincide with the inflammation, but in other models of inflammation transient adhesion mediated by members of the Selectin gene family was found to be essential before ICAM-1/beta 2 interactions could occur. We, therefore, wondered whether a similar sequence of initial transient adhesion followed by subsequent responses would be observed in hyperoxic lung inflammation. We, therefore, determined the effects of hyperoxia exposure on lung mRNA for P- and E-Selectin in mouse lungs. We found that there was no detectable mRNA for E-Selectin through 72 h of hyperoxia exposure by Northern blotting, but that mRNA for P-Selectin was detectable as early as 48 h after initiation of hyperoxia. To determine the location of P-Selectin upregulation we examined hyperoxia-exposed mouse lungs by in situ hybridization and found that the upregulation of P-Selectin at 48 h was localized to large muscularized vessels, at 72 h expression was detected in some medium size muscularized vessels, and at 96 h abundant expression was observed also on nonmuscularized small vessels. In conclusion, increases in mRNA for P-Selectin early in the course of hyperoxia exposure suggest that P-Selectin expression in hyperoxic lungs increases in parallel with upregulation of ICAM-1, leading to the accumulation of neutrophils in hyperoxic lungs, and that interventions targeting these two adhesion molecules may lead to a diminution in hyperoxic lung inflammation and lung injury.

Interleukin-1 expression during hyperoxic lung injury in the mouse.

An important component of the pathophysiologic response to hyperoxia (O2) is pulmonary inflammation, although the roles of specific inflammatory mediators during pulmonary O2 toxicity are not completely known. Interleukin-1 (IL-1) is an early inflammatory mediator and is sufficient to elicit many of the responses associated with acute injury. The IL-1 family comprises two bioactive proteins, IL-1alpha and IL-1beta, and their natural antagonist IL-1ra. Here we report studies of IL-1 regulation during hyperoxic lung injury in the adult mouse. When assayed by Northern blot, increases in IL-1beta mRNA were seen after 2 days of hyperoxia. In contrast, IL-1alpha mRNA was barely detectable before 4 days of hyperoxia. To further understand the cellular origin of IL-1beta expression in lungs, in situ hybridization and immunohistochemical analyses were performed. IL-1beta mRNA or protein was not detected in the lungs of unexposed animals. At 3 days, we observed the accumulation of IL-1beta transcripts in pulmonary interstitial macrophages and in a subset of neutrophils, and immunodetectable IL-1beta protein was co-localized in adjacent sections. At 4 days of exposure, IL-1beta transcripts were widespread in lung tissue, but many areas rich in IL-1beta mRNA were devoid of immunodetectable IL-1beta. However, it is not known whether increased synthesis of IL-1beta or the uncoupling of IL-1beta protein and mRNA accumulation has a role in pathophysiology of pulmonary O2 toxicity.

Early and persistent alterations in the expression of interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor alpha mRNA levels in fibrosis-resistant and sensitive mice after thoracic irradiation.

Fibrosis, characterized by the accumulation of collagen, is a consequence of a chronic inflammatory response. The purpose of this study was to determine if tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and IL-1 beta mRNA expression are altered acutely after irradiation, during the so-called "latent" phase of pulmonary injury, and to examine if these alterations persist through the development of pneumonitis and fibrosis. Further, we wished to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Fibrosis-sensitive (C57BL/6) and fibrosis-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by slot blotting and hybridized with radiolabeled cDNA probes encoding for TNF-alpha, IL-1 alpha and IL-1 beta. Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase. It was found that TNF-alpha mRNA levels were increased in C57BL/6 mice at days 1 and 7 postirradiation after 5 Gy and day 14 postirradiation after both 5 and 12.5 Gy, and IL-1 alpha mRNA levels were increased in C57BL/6 mice at days 56, 112 and 182 postirradiation after both 5 and 12.5 Gy, and IL-1 beta mRNA levels in the C3H/HeJ mice were increased at days 56 and 182 postirradiation after 12.5 Gy. In summary, these studies demonstrated early and persistent alterations in TNF-alpha, IL-1 alpha and IL-1 beta mRNA levels even at the lower dose (5 Gy). The temporal relationship between the elevation of these cytokines and the strain-dependent variation in fibrosis response suggests that IL-1 alpha and TNF-alpha contribute to the radiation-induced component of pulmonary fibrosis, whereas IL-1 beta may have a protective function.

Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis.

Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The purpose of this study was to determine if extracellular matrix protein and transforming growth factor beta mRNA expression are altered late in the course of pulmonary fibrosis after irradiation, and then to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Radiation-sensitive (C57BL/6) and radiation-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabeled cDNA probes for collagens I, III and IV, fibronectin, and transforming growth factor beta 1 and beta 3. Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase. Alterations in mRNA abundance were observed in the sensitive mice at all times, while levels in the resistant mice were unaffected until 26 weeks after irradiation. The relationship between extracellular matrix protein per se and increased mRNA abundance suggests that late matrix protein accumulation may be a function of gene expression. Differences in levels of transforming growth factor beta mRNA may lead to strain-dependent variation in fibrotic response and may also contribute to the radiation-induced component of pulmonary fibrosis.

Beneficial effect of coinfusing a lipid emulsion on venous patency.

The hypothesis that infused fat could prolong venous patency was tested in a paired crossover design. Parenterally fed newborn infants received, for a given level of energy, (60 vs 80 kcal/kg/day), two 6-day isocaloric and isonitrogenous (434 +/- 3.4 mg/kg/day, n = 32) regimens differing only by the fat intake (LF: 1.03 +/- 0.02, HF: 2.78 +/- 0.05 g/kg/day). Paired comparisons of osmolarities within isocaloric (60 or 80 kcal/kg/day) infusions showed that high fat regimens were associated with significantly lower osmolarities. A paired comparison of patency times showed that the drop in osmolarity produced by the high fat regimen at 60 kcal/kg/day led to a significantly longer venous patency time. The comparison of patency times between regimens (LF, 60 kcal/kg/day) and HF, 80 kcal/kg/day) with same osmolarities (702 mOsm/liter) and glucose intakes (11 g/kg/day) documented that the fat emulsion per se had a vascular protective effect. This observation demonstrates that the coinfusion of a lipid emulsion exerts a beneficial effect, whether biochemical or biophysical, on the vascular endothelium of peripheral veins.

Relative contribution of physical activity to neonatal oxygen consumption.

To explore the relevance of distinguishing between resting and global energy expenditure in newborn infants, oxygen consumption (VO2) was measured during extremes of physical activity in 17 parenterally fed newborn infants with a large range of body weights (1.0-3.4 kg) and gestational ages (28-41 weeks). Under constant nutrient intakes, each infant served as his/her own control when comparing VO2 during resting conditions and spontaneous intense physical activity, called exercise. VO2 was significantly correlated with body weight at rest (r = 0.96). But during intense activity, the better predictor of exercise-induced VO2 was body weight in the smaller infants (< 2.0 kg) and gestational age in the larger infants (> 35 weeks). The difference in VO2 between both levels of activity represented the oxygen cost of exercise, which decreased (P < 0.01) with body weight. For clinical purposes, the physical activity of low-birth-weight infants does not contribute substantially to their energy balance.

Induction of metallothionein and heme oxygenase in rats after inhalation of endotoxin.

Various stress proteins appear to play a role in injury and repair produced by inhaled pollutants. The present study examined the effect of inhaled endotoxin on the expression of the metallothionein and heme oxygenase genes. Rats were exposed to saline or endotoxin aerosols for 3 h and sacrificed up to 3 d following exposure. The significant induction of metallothionein mRNA in both the lung (fourfold increase) and liver (one-fold) were greatest at 3 h and returned to basal levels by 24 h after endotoxin exposure. Similarly, the increase in tissue metallothionein was greater in the lung. In situ hybridization in mice showed large increases in the relative abundance of metallothionein transcripts in epithelial cells of the conducting airways, in surrounding airway tissue, and in the nearby gas exchange region. While an endotoxin-induced significant increase in heme oxygenase mRNA followed a time course similar to that observed for metallo thionein, the relative magnitude was reversed for the lung and liver. Heme oxygenase mRNA was induced greater in the liver (twofold) than in the lung (60% above control). Our findings demonstrate that metallothionein and heme oxygenase are early response genes that are rapidly activated after inhalation of occupationally relevant concentrations of endotoxin.

Respiratory gas exchange in response to fat-free parenteral nutrition: a comparison after thoracic or abdominal surgery in newborn infants.

Thoracic surgery is known to cause a postoperative respiratory failure because of the mechanical problems following chest wall disruption and/or diaphragmatic dysfunction. This study was to verify whether the fat-free intravenous nutritional support of neonates who underwent thoracic surgery could lead to a CO2 production exceeding the patients' respiratory reserves. Respiratory gas exchange and alveolar ventilation were obtained by indirect calorimetry and continuous recordings of transcutaneous PO2 and PCO2. These noninvasive measurements were compared at the same age of 7 +/- 1 days between a group of 7 newborn infants (mean +/- SEM: 3.09 +/- 0.14 kg, 39 +/- 1 weeks) after thoracic surgery versus a group of 8 newborn infants (2.88 +/- 0.17 kg, 37 +/- 1 weeks) after abdominal surgery. The intravenous macronutrient support was the same between both groups: 14 g/kg/d of glucose, 2 g/kg/d of amino acids, 250 kJ/kg/d of energy. One week after surgery, the global metabolic rate (195 kJ/kg/d) was not increased, and comparable between both groups. We documented that early after thoracic surgery, the ventilatory compensation required to handle the CO2 production (6.7 +/- 0.2 mL/kg/min) associated with a positive energy balance (45 +/- 8 kJ/kg/d) was effective.

Pulmonary structural maturation and pulmonary artery remodeling after reversible fetal ovine tracheal occlusion in diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) is associated with thickened pulmonary arteries (PA) contributing to pulmonary hypertension. In the current study, the effects of antenatal glucocorticoids and reversible tracheal occlusion (TO) on PA structure were assessed in a hypoplastic lung model. METHODS: A left-sided CDH was created in fetal lambs at 80 days gestation, TO at 108 days, and release of the occlusion (TR) at 129 days. All were given 1 dose of maternal glucocorticoids at 135 days. At 136 days (term, 145 days), the fetus was delivered by cesarian section. CDH (n = 7), CDH + TO (n = 6), CDH + TO + TR (n = 6), and unoperated twin controls (n = 16) were compared. Outcome measurements were (1) lung growth, represented by lung weight to body weight ratio (LW/BW), (2) lung structural maturation, which is inversely proportional to mean terminal bronchiole density (MTBD), (3) PA medial and adventitial areas (square micrometers), (4) lung capillary load, which is the ratio of vessel surface area (SA) to tissue SA ratio. RESULTS: CDH lungs were hypoplastic with a low LW/BW and high MTBD. The small PAs (<75 microm) of CDH had an increased medial area, indicating increased muscle mass and an increased adventitial area. CDH + TO +/- TR increased LW/BW and achieved normal structural lung maturity with a low MTBD. Only CDH + TO thinned the PA medial area closer to control values. The adventitial area remained thick in CDH +/- TO +/- TR when compared with controls. All 4 groups had similar capillary load. CONCLUSIONS: TO may be especially important for PA remodeling in the latter part of gestation, because TR 1 week before delivery prevents thinning of the small PAs in CDH. The shaping achieved by TO in terms of lung growth, structural maturity, and pulmonary artery medial area thinning may prove beneficial in lessening the severity of the associated pulmonary hypertension in CDH.

The plug-unplug sequence: an important step to achieve type II pneumocyte maturation in the fetal lamb model.

PURPOSE: The purpose of this study was to test the hypothesis that tracheal obstruction (plugging) in the fetal lamb model leads to a decrease in the absolute number of type II pneumocytes and that reversing the obstruction before birth (unplugging), allows the type II cells to recover while maintaining the beneficial effect on lung growth. METHODS: Nine time-dated pregnant ewes (term, 145 days), carrying 17 fetuses, were used in this surgical trial. The fetuses were divided into three experimental groups: group A underwent plugging at 93 days gestation, followed by unplugging at 110 days; group B animals had tracheal ligation at 93 days and group C consisted of unoperated controls. All fetuses were delivered by cesarean section at 136 days' gestation. The fetal trachea was obstructed with the tracheoscopically placed detachable balloon described by our group. Unplugging was performed by needle puncture of the balloon under tracheoscopic vision. Outcome measurements consisted of lung-to-body-weight ratio (LWBR), lung morphometry (mean terminal bronchial density [MTBD] and linear intercept [Lm]), and assessment of the number of type II pneumocytes. The latter was determined by in situ hybridization to the mRNA of surfactant protein-C, which is exclusively produced by type II cells. Statistics were calculated using a two-tailed unpaired t test and P less than .05 is considered significant. RESULTS: Seventeen animals are included in the results. All of them had lung samples analyzed for lung morphometry, whereas for type II cells analysis, three animals were studied in each group. Morphometric analyses were consistent with pulmonary hyperplasia for group B, whereas group A lungs showed more histological maturity than group C albeit not as marked as group B. In group A, there was a similar number of type II cells to that observed in group C (53.2 +/- 3.9 v 55.9 +/- 4.0, P = .66). However, for group B animals, the number of type II pneumocytes was markedly decreased compared with controls (4.7 +/- 0.1 v 55.9 +/- 4, P = .0003). CONCLUSIONS: The authors conclude that tracheal ligation until birth, although inducing pulmonary hyperplasia, significantly decreases the number of type II pneumocytes in the alveoli. After a temporary 15-day occlusion initiated at 95 days' gestation, there is complete normalization of the density of type II cells. These results bear importance on the duration of PLUG to treat the pulmonary hypoplasia seen in congenital diaphragmatic hernia. Temporary tracheal obstruction now needs to be tested in a hypoplastic lung model.

Surfactant levels after reversible tracheal occlusion and prenatal steroids in experimental diaphragmatic hernia.

BACKGROUND/PURPOSE: In normal lungs, fetal tracheal occlusion (TO) induces lung growth but decreases the number of type II cells; this is remedied if TO is released (TR) before delivery. In the current study, the effects of TO with or without TR on pulmonary structure and surfactant were assessed in the ovine model in which lung hypoplasia was induced by creation of a diaphragmatic hernia (CDH). METHODS: A left-sided CDH was created in fetal lambs at 80 days gestation; TO was done at 108 days; and TR at 129 days. All ewes were given 1 dose of glucocorticoids at 135 days. At 136 days, the fetus was delivered. Lung weight to body weight ratio, mean terminal bronchiole density, type II cell density, bronchoalveolar lavage fluid (BAL) phosphatidylcholine (PC), BAL surfactant protein A (SP-A) and B (SP-B), and lung tissue SP-A and SP-B were assessed in CDH, CDH with TO, CDH with TO and TR, and controls. RESULTS: CDH lungs were hypoplastic and structurally immature, but had increased type II cell density. TO with or without TR caused lung growth with normalization of lung parenchymal architecture and type II cell density. Although the BAL SP-A and BAL SP-B were similar in all 4 groups, the BAL PC was low in CDH with or without TO or TR. Also, lung tissue SP-B levels were low in CDH with or without TO or TR. However, lung tissue SP-A levels were normal in CDH, but low in CDH with TO with or without TR. CONCLUSIONS: Despite the finding that lung morphology was improved in CDH with TO with or without TR animals, surfactant content and composition remained abnormal. Although surfactant secreted early by the fetus into alveolar spaces contained normal levels of BAL SP-A and BAL SP-B, the low levels of BAL PC and low lung tissue stores of SP-B indicate that these experimental lambs may experience respiratory insufficiency soon after birth. This implies that prophylactic surfactant at birth might be beneficial for CDH.

The effects of tracheal occlusion and release on type II pneumocytes in fetal lambs.

UNLABELLED: Fetal tracheal occlusion (TO) has been shown to lead to lung hyperplasia in various animal models, and this procedure has already been carried out in human fetuses with congenital diaphragmatic hernia (CDH). However, the authors previously showed that TO caused a decrease in type II pneumocytes. PURPOSE: The aim of this study is to examine the effects of TO and release on type II pneumocytes. METHOD: To was carried out with a Swan Ganz or Fogarty catheter in fetal sheep at 116 to 118 days of gestation. TO was maintained for 2 weeks followed by deflation of the balloon for 1 week before delivery, in group 1; in group 2, TO was maintained for 19 days and released 2 days before delivery. Group 3 consisted of previously reported animals who had TO maintained until birth. Unoperated twins served as controls. All specimens were analyzed using the surfactant protein C (SP-C) mRNA as a specific marker for type II pneumocytes. We used Northern Blot and in situ hybridization techniques to quantify total SP-C and the density of type II cells. Electron microscopy (EM) was also used to evaluate and quantitate type II cells. RESULTS: TO resulted in significant lung growth in all groups. In situ hybridization and Northern Blot analysis showed that there was a complete recovery of type II cells in group 1 versus controls. Quantitative EM analysis confirmed these findings. In group 2 the number of type II cells was decreased but there was an increase in SP-C content per type II cell versus group 3. CONCLUSION: Lung growth after TO appears to occur at the expense of type II cell differentiation. This effect is reversible with the release of TO before birth in this animal model.

Energy expenditure and severity of respiratory disease in very low birth weight infants receiving long-term ventilatory support.

We attempted to determine whether the hypermetabolism of infants with bronchopulmonary dysplasia was detectable during assisted ventilation. Respiratory gas exchange variables were measured with a metabolic gas monitor in 10 infants under similar nutritional conditions. Oxygen consumption increased linearly with the need for ventilatory support (R2 = 0.75), as documented by the ventilatory index.

Influence of energy substrates on respiratory gas exchange during conventional mechanical ventilation of preterm infants.

OBJECTIVE: The purpose of this study was to determine the optimal parenteral feeding regimen for infants with compromised respiratory function. METHODS: We studied the influence of varying the source of energy on respiratory gas exchange in 10 infants who were supported by mechanical ventilation and who received intravenous feedings. Two isoenergetic parenteral regimens were infused consecutively; the level of fat intake was varied inversely with that of glucose. Under similar ventilator settings, transcutaneous partial pressures of oxygen and carbon dioxide, as well as indirect calorimetry were measured during each regimen. RESULTS: Despite the higher carbon dioxide production during the glucose-rich regimen (8.9 +/- 0.7 vs 7.9 +/- 0.4 ml/kg per minute, p < 0.05 by analysis of variance), transcutaneous partial pressure of carbon dioxide remained unaffected, suggesting ventilatory compensation as documented by the increased (p < 0.002) alveolar ventilation. This was not associated with a detectable rise in oxygen consumption, but with a significant change in partial pressure of oxygen (77 +/- 5 vs 66 +/- 3 mm Hg, p < 0.05). CONCLUSIONS: Ventilator-dependent infants with early and mild bronchopulmonary dysplasia, who receive intravenous feedings of a moderate load of glucose-based energy, can compensate for enhanced carbon dioxide production by increasing their respiratory drive, with a beneficial effect on oxygenation compared with that observed when energy is derived from lipid-based solutions.