Moderate prenatal ethanol exposure in the rat promotes kidney cell apoptosis, nephron deficits, and sex-specific kidney dysfunction in adult offspring.

Alcohol during pregnancy can impair fetal development and result in offspring with neurodevelopmental deficits. Less is known about how low to moderate alcohol exposure can affect other organs, such as the kidney. Here, the effects of moderate ethanol exposure throughout pregnancy on kidney development were examined using a rat model. Rats were fed a liquid diet containing 6% ethanol (vol/vol) or control (0% ethanol) throughout pregnancy. Kidneys were collected at embryonic day (E) 20 or postnatal day (PN) 30 and total glomerular (nephron) number determined using unbiased stereology. Kidney function was examined in offspring at 8 and 19 months. At E20, fetuses exposed to ethanol had fewer nephrons with increased apoptosis. Alcohol exposure caused kidney dysregulation of pro- (Bax) and anti- (Bcl-2) apoptotic factors, and reduced expression of the cell proliferation marker, Ki67. Prenatal alcohol decreased expression of Gdnf and Tgfb1, important regulators of branching morphogenesis, in male fetuses. At PN30, kidney volume and nephron number were lower in offspring exposed to prenatal alcohol. Urine flow and osmolality were normal in offspring exposed to alcohol however sodium excretion tended to be lower in females prenatally exposed to alcohol. Findings suggest exposure to moderate levels of alcohol during pregnancy results in impaired kidney development and leads to a permanent nephron deficit. Although the impact on adult kidney function was relatively minor, these data highlight that even at moderate levels, alcohol consumption during pregnancy can have deleterious long-term outcomes and should be avoided.

Low-dose maternal alcohol consumption: effects in the hearts of offspring in early life and adulthood.

High alcohol consumption during pregnancy leads to deleterious effects on fetal cardiac structure and it also affects cardiomyocyte growth and maturation. This study aimed to determine whether low levels of maternal alcohol consumption are also detrimental to cardiomyocyte and cardiac growth in the early life of offspring and whether cardiac structure and function in adulthood is affected. Pregnant Sprague-Dawley rat dams were fed a control or 6% (volume/volume) liquid-based ethanol supplemented (isocaloric) diet throughout gestation. At embryonic day 20, the expression of genes involved in cardiac development was analyzed using Real-time PCR. At postnatal day 30, cardiomyocyte number, size, and nuclearity in the left ventricle (LV) were determined stereologically. In 8-month-old offspring, LV fibrosis and cardiac function (by echocardiography) were examined. Maternal ethanol consumption did not alter gene expression of the cardiac growth factors in the fetus or cardiomyocyte number in weanling offspring. However, at 8 months, there were significant increases in LV anterior and posterior wall thickness during diastole in ethanol-exposed offspring (P = 0.037 and P = 0.024, respectively), indicative of left ventricular hypertrophy; this was accompanied by a significant increase in fibrosis. Additionally, maximal aortic flow velocity was significantly decreased in ethanol-exposed offspring (P = 0.035). In conclusion, although there were no detectable early-life differences in cardiac and cardiomyocyte growth in animals exposed to a chronic low dose of ethanol during gestation, there were clearly deleterious outcomes by adulthood. This suggests that even relatively low doses of alcohol consumed during pregnancy can be detrimental to long-term cardiac health in the offspring.

The effect of low-to-moderate-dose ethanol consumption on rat mammary gland structure and function and early postnatal growth of offspring.

High levels of alcohol consumption during pregnancy can lead to growth deficits in early postnatal life. However, the effects of low-to-moderate alcohol consumption during pregnancy are less clearly defined. The aim of this study was to determine whether low-to-moderate ethanol (EtOH) consumption throughout pregnancy in the rat alters maternal mammary gland morphology and milk protein levels, thereby affecting lactation and the growth of pups after birth. Sprague-Dawley rats were fed an ad libitum liquid diet ± 6% vol/vol EtOH throughout pregnancy. Mammary glands from dams were collected at embryonic day (E) 20 or postnatal day (PN) 1, and expression of milk proteins (α-lactalbumin, ß-casein, and whey acidic protein) was examined. In addition, relative amounts of alveoli, lactiferous ducts, adipose tissue, and blood vessels were determined at PN1. A subset of rats gave birth, and offspring growth and milk intake were recorded. Mammary gland weight was unaltered by EtOH, and stereological analysis showed no differences in gland structure compared with control. Although there were no significant changes in mammary gland gene expression at the RNA level, protein levels of α-lactalbumin were increased and whey acidic protein were decreased by EtOH. Offspring of EtOH-fed dams consumed less milk than controls in the lactational period; however, this did not alter their early postnatal growth. Overall, it appears that low-to-moderate-dose prenatal EtOH exposure does not significantly alter mammary gland development but may alter the composition of the various proteins found within the milk in a manner that maintains overall pup growth.

Impact of low dose prenatal ethanol exposure on glucose homeostasis in Sprague-Dawley rats aged up to eight months.

Excessive exposure to alcohol prenatally has a myriad of detrimental effects on the health and well-being of the offspring. It is unknown whether chronic low-moderate exposure of alcohol prenatally has similar and lasting effects on the adult offspring's health. Using our recently developed Sprague-Dawley rat model of 6% chronic prenatal ethanol exposure, this study aimed to determine if this modest level of exposure adversely affects glucose homeostasis in male and female offspring aged up to eight months. Plasma glucose concentrations were measured in late fetal and postnatal life. The pancreas of 30 day old offspring was analysed for ß-cell mass. Glucose handling and insulin action was measured at four months using an intraperitoneal glucose tolerance test and insulin challenge, respectively. Body composition and metabolic gene expression were measured at eight months. Despite normoglycaemia in ethanol consuming dams, ethanol-exposed fetuses were hypoglycaemic at embryonic day 20. Ethanol-exposed offspring were normoglycaemic and normoinsulinaemic under basal fasting conditions and had normal pancreatic ß-cell mass at postnatal day 30. However, during a glucose tolerance test, male ethanol-exposed offspring were hyperinsulinaemic with increased first phase insulin secretion. Female ethanol-exposed offspring displayed enhanced glucose clearance during an insulin challenge. Body composition and hepatic, muscle and adipose tissue metabolic gene expression levels at eight months were not altered by prenatal ethanol exposure. Low-moderate chronic prenatal ethanol exposure has subtle, sex specific effects on glucose homeostasis in the young adult rat. As aging is associated with glucose dysregulation, further studies will clarify the long lasting effects of prenatal ethanol exposure.

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.

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-, ß-, γ-subunits) and Na(+)-K(+)-ATPase (α-, ß-, 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.

The anti-inflammatory agent N-acetyl cysteine exacerbates endotoxin-induced hypoxemia and hypotension and induces polycythemia in the ovine fetus.

BACKGROUND: Lipopolysaccharide (LPS) delivered acutely to the ovine fetus induces cerebral white matter injury and brain inflammation. N-acetyl cysteine (NAC) is potentially neuroprotective as it blocks the production of inflammatory cytokines and increases glutathione levels; however, it is unknown whether NAC affects the physiological status of the fetus already exposed to an inflammatory environment. OBJECTIVES: Our objective was to determine whether NAC influences the physiological effects of LPS exposure in the ovine fetus. METHODS: Catheterized fetal sheep underwent one of four treatments (saline, n = 6; LPS, n = 6; LPS + NAC, n = 6; NAC, n = 3) on 5 consecutive days from 95 days of gestation (term approximately 147 days). Fetal arterial pressure and heart rate were recorded and blood samples collected. RESULTS: LPS administration resulted in fetal hypoxemia and hypotension; simultaneous treatment with NAC exacerbated these effects and induced polycythemia. NAC treatment alone had no effect on the fetus. CONCLUSION: In the presence of LPS, NAC compromises fetal physiological status, suggesting that it may not be a suitable antenatal treatment for a fetus with evidence of inflammation.

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

BACKGROUND: Bronchopulmonary 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. METHODS: To identify early markers of VILI, preterm lambs (132 d gestational age; GA, term approximately 147 d) were resuscitated with an injurious ventilation strategy (V(T) 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 V(T) 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. RESULTS: CTGF, CYR61 and EGR1 lung mRNA levels were increased approximately 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- , 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. CONCLUSION: CTGF, 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.

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. (c) 2008 Wiley-Liss, Inc.

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.

Does moderate preterm birth lead to altered arterial pressure? Studies in sheep.

1. Low birth weight (LBW) is associated with an increased risk of cardiovascular disease. Preterm birth is a major determinant of LBW and has been shown to result in elevated arterial pressure (AP) in humans, but few studies have investigated the effects of preterm birth in the absence of potentially confounding factors. Our aim was to determine whether moderately preterm birth per se alters the postnatal development of AP in lambs. 2. Preterm lambs were delivered approximately 14 days before term (i.e. approximately 133 days of gestation); controls were born at term (approximately 147 days). Mean arterial pressure (MAP), heart rate (HR), blood composition and indices of growth were measured at 4 and 8 weeks post term-equivalent age (PTEA). We also studied a separate cohort of preterm and term sheep as young adults (approximately 1.1 years). 3. Preterm lambs had significantly lower birth weights than term lambs, but bodyweights were not significantly different by Day 12 PTEA. In addition, MAP, HR and most blood variables did not differ between term and preterm lambs at 4 or 8 weeks PTEA. Preterm birth per se did not alter MAP or HR in young adult sheep. 4. Low birth weight due to preterm birth does not result in an altered AP during early postnatal life or at maturity. Moderate intrauterine growth restriction (IUGR) due to twinning, which further reduces birth weight, does not affect MAP in preterm lambs. Other factors, such as the degree of prematurity or IUGR, exposure to corticosteroids or postnatal nutrition, may be important in the later development of elevated AP.

Spontaneously occurring differences in fetal weight do not affect blood pressure, the hypothalamic-pituitary-adrenal axis or the renin-angiotensin system in the late-gestation ovine fetus.

Fetal growth restriction (FGR) has been associated with an increased incidence of cardiovascular disease in adult life. Animal models of restricted fetal growth often cause FGR over discrete periods of gestation and hence may not be applicable to individuals with low birthweight but who are not clinically growth-restricted. Our aim was to determine whether spontaneously occurring differences in fetal growth influence the functional development of the hypothalamic-pituitary-adrenal (HPA) axis or the renin-angiotensin system (RAS), both of which are involved in arterial pressure regulation. Using sheep, arterial pressure and heart rate were monitored in chronically catheterised singleton and twin fetuses at 130, 134 and 137 days of gestation (term approximately 147 days). Fetuses were challenged, at different times, with exogenous angiotensin (Ang) II, combined administration of arginine vasopressin and corticotrophin releasing hormone (AVP+CRH) and adrenocorticotrophic hormone (ACTH); fetal cardiovascular responses and circulating cortisol concentrations were measured. In all fetuses Ang II and AVP+CRH altered cardiovascular function (increase in mean arterial pressure and decrease in heart rate); both AVP+CRH and ACTH increased circulating cortisol concentrations. Responses were not related to fetal bodyweight. We conclude that naturally occurring differences in growth do not influence the development of the HPA axis or RAS function in fetal sheep.

Effects of antenatal corticosteroid treatment on pulmonary ventilation and circulation in neonatal lambs with hypoplastic lungs.

Our aim was to determine whether antenatal corticosteroids improve perinatal adaptation of the pulmonary circulation in lambs with lung hypoplasia (LH). LH was induced in 12 ovine fetuses between 105 and 140 days gestation (term approximately 147 days); in 6 of these the ewe was given a single dose of betamethasone (11.4 mg im) 24 hr before delivery (LH + B). All lambs, including a control group (n = 6), were delivered at approximately 140 days and ventilated for 2 hr during which arterial pressures, pulmonary blood flow (PBF), and ventilating pressure and flow were recorded. During ventilation, respiratory system compliance was lower in both LH + B and LH groups than in controls. Pulmonary vascular resistance (PVR) was lower in LH + B lambs than in LH lambs and similar to controls; PBF was reduced in LH lambs but was restored to control levels by betamethasone. The mean density of small arteries of LH + B lambs was similar to that of LH lambs (P = 0.06) and lower than in controls; the thickness of the media of small pulmonary arteries from LH + B lambs was similar to that in LH lambs and thicker than in controls. VEGF mRNA levels were not different between groups. PDGF mRNA levels in LH + B lambs were higher than in LH lambs; a similar trend (P = 0.06) was seen for PECAM-1. SP-C mRNA levels were greater in both LH and LH + B lambs than in controls. Effects of betamethasone were greater on indices of pulmonary circulation than ventilation. We conclude that a single dose of maternal betamethasone 24 hr prior to birth has significant favorable effects on the postnatal adaptation of the pulmonary circulation in lambs with LH.

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.

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 approximately 125 d gestation (term approximately 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 H(2)O 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 H(2)O of PEEP died from pneumothoraces; all others survived without pneumothoraces. Oxygenation was significantly improved by 8 and 12 cm H(2)O of PEEP compared with 0 and 4 cm H(2)O of PEEP. Lambs with 0 PEEP did not oxygenate adequately. The compliance of the respiratory system was significantly higher at 4 and 8 cm H(2)O 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 H(2)O 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.