Megan E. Probyn

Positive End Expiratory Pressure during Resuscitation of Premature Lambs Rapidly Improves Blood Gases without Adversely Affecting Arterial Pressure

Positive end expiratory pressure (PEEP) is important for neonatal ventilation but is not considered in guidelines for resuscitation. Our aim was to investigate the effects of PEEP on cardiorespiratory parameters during resuscitation of very premature lambs delivered by hysterotomy at ∼125 d gestation (term ∼147 d). Before delivery, they were intubated and lung fluid was drained. Immediately after delivery, they were ventilated with a Dräger Babylog plus ventilator in volume guarantee mode with a tidal volume of 5 mL/kg. Lambs were randomized to receive 0, 4, 8, or 12 cm H2O of PEEP. They were ventilated for a 15-min resuscitation period followed by 2 h of stabilization at the same PEEP. Tidal volume, peak inspiratory pressure, PEEP, arterial pressure, oxygen saturation, and blood gases were measured regularly, and respiratory system compliance and alveolar/ arterial oxygen differences were calculated. Lambs that received 12 cm H2O of PEEP died from pneumothoraces; all others survived without pneumothoraces. Oxygenation was significantly improved by 8 and 12 cm H2O of PEEP compared with 0 and 4 cm H2O of PEEP. Lambs with 0 PEEP did not oxygenate adequately. The compliance of the respiratory system was significantly higher at 4 and 8 cm H2O of PEEP than at 0 PEEP. There were no significant differences in partial pressure of carbon dioxide in arterial blood between groups. Arterial pressure was highest with 8 cm H2O of PEEP, and there was no cardiorespiratory compromise at any level of PEEP. Applying PEEP during resuscitation of very premature infants might be advantageous and merits further investigation.

Early biomarkers and potential mediators of ventilation-induced lung injury in very preterm lambs

BackgroundBronchopulmonary dysplasia (BPD) is closely associated with ventilator-induced lung injury (VILI) in very preterm infants. The greatest risk of VILI may be in the immediate period after birth, when the lungs are surfactant deficient, still partially filled with liquid and not uniformly aerated. However, there have been very few studies that have examined this immediate post-birth period and identified the initial injury-related pathways that are activated. We aimed to determine if the early response genes; connective tissue growth factor (CTGF), cysteine rich-61 (CYR61) and early growth response 1 (EGR1), were rapidly induced by VILI in preterm lambs and whether ventilation with different tidal volumes caused different inflammatory cytokine and early response gene expression.MethodsTo identify early markers of VILI, preterm lambs (132 d gestational age; GA, term ~147 d) were resuscitated with an injurious ventilation strategy (VT 20 mL/kg for 15 min) then gently ventilated (5 mL/kg) for 15, 30, 60 or 120 min (n = 4 in each). To determine if early response genes and inflammatory cytokines were differentially regulated by different ventilation strategies, separate groups of preterm lambs (125 d GA; n = 5 in each) were ventilated from birth with a VT of 5 (VG5) or 10 mL/kg (VG10) for 135 minutes. Lung gene expression levels were compared to levels prior to ventilation in age-matched control fetuses.ResultsCTGF, CYR61 and EGR1 lung mRNA levels were increased ~25, 50 and 120-fold respectively (p < 0.05), within 30 minutes of injurious ventilation. VG5 and VG10 caused significant increases in CTGF, CYR61, EGR1, IL1-β, IL-6 and IL-8 mRNA levels compared to control levels. CTGF, CYR61, IL-6 and IL-8 expression levels were higher in VG10 than VG5 lambs; although only the IL-6 and CYR61 mRNA levels reached significance.ConclusionCTGF, CYR61 and EGR1 may be novel early markers of lung injury and mechanical ventilation from birth using relatively low tidal volumes may be less injurious than using higher tidal volumes.

Developmental programming of a reduced nephron endowment: more than just a baby's birth weight

The risk of developing many adult-onset diseases, including hypertension, type 2 diabetes, and renal disease, is increased in low-birth-weight individuals. A potential underlying mechanism contributing to the onset of these diseases is the formation of a low nephron endowment during development. Evidence from the human, as well as many experimental animal models, has shown a strong association between low birth weight and a reduced nephron endowment. However, other animal models, particularly those in which the mother is exposed to elevated glucocorticoids for a short period, have shown a 20-40% reduction in nephron endowment without discernible changes in the birth weight of offspring. Such findings emphasize that a low birth weight is one, but certainly not the only, predictor of nephron endowment and suggests reduced nephron endowment and risk of developing adult-onset disease, even among normal-birth-weight individuals. Recognition of the dissociation between birth weight and nephron endowment is important for future studies aimed at elucidating the role of a reduced nephron endowment in the developmental programming of adult disease.

Erythropoietin Is Neuroprotective in a Preterm Ovine Model of Endotoxin-Induced Brain Injury

Intrauterine infection and inflammation have been linked to preterm birth and brain damage. We hypothesized that recombinant human erythropoietin (rhEPO) would ameliorate brain damage in anovine model of fetal inflammation. At 107 ± 1 day of gestational age (DGA), chronically catheterized fetal sheep received on 3 consecutive days 1) an intravenous bolus dose of lipopolysaccharide ([LPS] ∼0.9 &mgr;g/kg; n = 8); 2) an intravenous bolus dose of LPS, followed at 1 hour by 5,000 IU/kg of rhEPO (LPS + rhEPO, n = 8); or 3) rhEPO (n = 5). Untreated fetuses (n = 8) served as controls. Fetal physiological parameters were monitored, and fetal brains and optic nerves were histologically examined at 116 ± 1 DGA. Exposure to LPS, but not to rhEPO alone or saline, resulted in fetal hypoxemia, hypotension (p < 0.05), brain damage, including white matter injury, and reductions in numbers of myelinating oligodendrocytes in the corticospinal tract and myelinated axons in the optic nerve (p < 0.05 for both). Treatment of LPS-exposed fetuses with rhEPO did not alter the physiological effects of LPS but reduced brain injury and was beneficial to myelination in the corticospinal tract and the optic nerve. This is the first study in a long-gestation species to demonstrate the neuroprotective potential of rhEPO in reducing fetal brain and optic nerve injury after LPS exposure.

Prenatal glucocorticoid exposure in the sheep alters renal development in utero: implications for adult renal function and blood pressure control

Treatment of the pregnant ewe with glucocorticoids early in pregnancy results in offspring with hypertension. This study examined whether glucocorticoids can reduce nephron formation or alter gene expression for sodium channels in the late gestation fetus. Sodium channel expression was also examined in 2-mo-old lambs, while arterial pressure and renal function was examined in adult female offspring before and during 6 wk of increased dietary salt intake. Pregnant ewes were treated with saline (SAL), dexamethasone (DEX; 0.48 mg/h) or cortisol (CORT; 5 mg/h) over days 26-28 of gestation (term = 150 days). At 140 days of gestation, glomerular number in CORT and DEX animals was 40 and 25% less, respectively, compared with SAL controls. Real-time PCR showed greater gene expression for the epithelial sodium channel (α-, β-, γ-subunits) and Na(+)-K(+)-ATPase (α-, β-, γ-subunits) in both the DEX and CORT group fetal kidneys compared with the SAL group with some of these changes persisting in 2-mo-old female offspring. In adulthood, sheep treated with dexamethasone or cortisol in utero had elevated arterial pressure and an apparent increase in single nephron glomerular filtration rate, but global renal hemodynamics and excretory function were normal and arterial pressure was not salt sensitive. Our findings show that the nephron-deficit in sheep exposed to glucocorticoids in utero is acquired before birth, so it is a potential cause, rather than a consequence, of their elevated arterial pressure in adulthood. Upregulation of sodium channels in these animals could provide a mechanistic link to sustained increases in arterial pressure in cortisol- and dexamethasone-exposed sheep, since it would be expected to promote salt and water retention during the postnatal period.

Effects of tidal volume and positive end-expiratory pressure during resuscitation of very premature lambs

BACKGROUND Guidelines recommend neonatal resuscitation without controlling tidal volume or positive end-expiratory pressure (PEEP). However, these may improve gas exchange, lung volume and outcome. AIM To investigate resuscitation of very premature lambs with a Laerdal bag without PEEP versus volume guarantee ventilation with PEEP. METHODS Anaesthetized lambs (n=20) delivered at 125 d gestation were randomized to three groups receiving 15 min resuscitation: (1) Laerdal bag and no PEEP; (2) ventilation with a tidal volume of 5 ml/kg and 8 cm H(2)O PEEP; (3) ventilation with 10 ml/kg and 8 cm H(2)O PEEP. They were then all ventilated for 2 h with tidal volumes of 5 or 10 ml/kg, and 8 cm H(2)O PEEP. Ventilation parameters and blood gases were recorded. RESULTS Different tidal volumes affected PaCO(2) within minutes, with 10 ml/kg causing severe hypocarbia. PEEP had little effect on PaCO(2). Oxygenation improved significantly with PEEP of 8 cm H(2)O, irrespective of tidal volume. CONCLUSION Very premature lambs can be resuscitated effectively using volume-guarantee ventilation and PEEP. Tidal volumes affected PaCO(2) within minutes but had little effect on oxygenation. PEEP halved the oxygen requirement compared with no PEEP. Resuscitating premature babies with controlled tidal volumes and PEEP might improve their outcome.

Effect of increased lung expansion on lung growth and development near midgestation in fetal sheep.

Obstruction of the fetal trachea is a potent stimulus for fetal lung growth and may have therapeutic potential in human fetuses with lung hypoplasia. However, the effects of increased lung expansion on lung development near midgestation, which is the preferred timing for fetal intervention, have not been well studied. Our aim was to determine the effects of increased lung expansion on lung development at 75–90 d of gestation in fetal sheep. In three groups of fetuses (n = 4 for each), the trachea was occluded for either 10 [10-d tracheal occlusion (TO) group] or 15 d (15-d TO group) or left intact (control fetuses). TO for both 10 and 15 d caused fetal hydrops, resulting in significantly increased fetal body weights. Both periods of TO significantly increased total lung DNA contents from 99.8 ± 10.1 to 246.0 ± 5.3 and 246.9 ± 48.7 mg in 10- and 15-d TO fetuses, respectively. TO for 10 and 15 d also increased airspace diameter, although the percentage of lung occupied by airspace was not increased in 10-d TO fetuses due to large increases in interairway distances; this resulted from a large increase in mesenchymal tissue. The interairway distances at 15 d of TO were reduced compared with the 10-d value but were still ∼30% larger than control values. We conclude that TO at <90 d of gestation in fetal sheep induces a greater increase in lung tissue growth than later in gestation but also causes fetal hydrops and produces changes in lung structure that are not compatible with efficient gas exchange. Thus, increased lung expansion at a similar stage of development in human fetuses is unlikely to induce changes in lung development that would facilitate gas exchange after birth.

The Influence of Naturally Occurring Differences in Birthweight on Ventricular Cardiomyocyte Number in Sheep

In most species including man, cardiomyocytes cease proliferating soon after birth when they become terminally differentiated. A reduced complement of cardiomyocytes in infancy may adversely impact on the function and adaptive capabilities of the heart in later life. Low birthweight is associated with an increased risk of heart disease in adults, but little is known about its effect on the number of cardiomyocytes. Using naturally occurring differences in birthweight, our aim was to determine the effect of birthweight on cardiomyocyte number in postnatal lambs. At 9 weeks after term birth, when the final number of cardiomyocytes is considered to be established, hearts were collected at necropsy from seven singleton and seven twin lambs. Hearts were perfusion‐fixed, and tissue samples were systematically taken from the left ventricle plus intraventricular septum (LV+S) and the right ventricle (RV). The number of cardiomyocyte nuclei was estimated using an unbiased optical disector–fractionator stereological technique, and the total number of cardiomyocytes was determined. Weights of the total heart, LV+S and RV were significantly related to both birthweight and necropsy weight. In the LV+S but not the RV, cardiomyocyte number was significantly and directly related to heart tissue weight, birthweight, and necropsy weight. We conclude that the final number of cardiomyocytes in the LV+S is related to prenatal and early postnatal growth, and is proportionate to the weight of heart tissue. A low cardiomyocyte number in the LV+S following restricted fetal growth may contribute to the increased incidence of heart disease in adults born with low birthweight. Anat Rec, 2009.

Lung Parenchyma at Maturity Is Influenced by Postnatal Growth but Not by Moderate Preterm Birth in Sheep

Background: We have recently shown that moderate preterm birth, in the absence of respiratory support, altered the structure of lung parenchyma in young lambs, but the long-term effects are unknown. Objectives: To determine whether structural changes persist to maturity, and whether postnatal growth affects lung structure at maturity in sheep. Methods: At approximately 1.2 years after birth, lung parenchyma of sheep born 14 days before term (n = 7) was stereologically compared with that of controls born at term (n = 8, term approx. 146 days). Results: Preterm birth per se had no significant effect on lung volume, alveolar number and size, and thicknesses of the alveolar walls and blood-gas barrier. After combining the preterm and term groups, we examined the effects of postnatal growth rates on lung parenchyma. Slower-growing sheep (SG; n = 7: 4 preterm, 3 term) were compared with faster-growing sheep (FG; n = 8: 3 preterm, 5 term). At approximately 1.2 years, the right lung volume, relative to body weight, was significantly lower in SG than FG sheep (p < 0.05) and alveolar number was significantly lower by approximately 44%. The total alveolar internal surface area of the right lung of SG sheep was 38% smaller than in FG sheep; it was also significantly lower when related to both lung and body weight. Conclusions: Our data suggest that moderate preterm birth does not cause persistent alterations in lung parenchyma. However, slow postnatal growth in low-birth-weight sheep results in smaller lungs with fewer alveoli and a lower alveolar surface area relative to body weight.

A rodent model of low- to moderate-dose ethanol consumption during pregnancy: patterns of ethanol consumption and effects on fetal and offspring growth

It is unknown whether low to moderate maternal alcohol consumption adversely affects postnatal health. The aim of the present study was to develop a rodent model of low-moderate-dose prenatal ethanol (EtOH) exposure. Sprague-Dawley rats were fed a liquid diet with or without 6% v/v EtOH throughout gestation and the pattern of dietary consumption determined. Fetal bodyweights and hepatic alcohol-metabolising gene expression were measured on embryonic Day (E) 20 and offspring growth studied until 1 year. At E8 the plasma EtOH concentration was 0.03%. There was little difference in dietary consumption between the two treatment groups. At E20, EtOH-exposed fetuses were significantly lighter than controls and had significantly decreased ADH4 and increased CYP2E1 gene expression. Offspring killed on postnatal Day (PN) 30 did not exhibit any growth deficits. Longitudinal repeated measures of offspring growth demonstrated slower growth in males from EtOH-fed dams between 7 and 12 months of age; a cohort of male pups killed at 8 months of age had a reduced crown-rump length and kidney weight. In conclusion, a liquid diet of 6% v/v EtOH fed to pregnant dams throughout gestation caused a 3-8% reduction in fetal growth and brain sparing, with growth differences observed in male offspring later in life. This model will be useful for future studies on the effects of low-moderate EtOH on the developmental origins of health and disease.