Dino A. Giussani

Afferent and efferent components of the cardiovascular reflex responses to acute hypoxia in term fetal sheep.

1. We studied the effects of acute isocapnic hypoxia on arterial and central venous pressures, carotid and femoral blood flows and heart rate in intact and carotid denervated fetal sheep between 118 and 125 days gestation, after pre‐treatment with either saline, atropine or phentolamine. Electrocortical activity (ECoG) and the incidence of fetal breathing movements (FBM) were also compared between intact and carotid denervated fetuses. 2. There were no significant differences between intact and denervated fetuses in any variable measured during normoxia. Soon after the onset of hypoxia a marked bradycardia occurred in intact, but not in denervated fetuses. Femoral blood flow and femoral vascular resistance (perfusion pressure/femoral blood flow) increased in intact, but not in denervated fetuses. Carotid blood flow increased in both groups of fetuses during hypoxia, but carotid vascular resistance did not change. During hypoxia, the incidence of FBM and low‐voltage ECoG was similarly reduced in both groups of fetuses. 3. Atropine produced a rise in fetal heart rate during the control period in intact but not in denervated fetuses. At the onset of hypoxia atropine prevented the initial bradycardia seen in intact fetuses. In denervated fetuses a further increase in heart rate occurred throughout the hypoxia. 4. All denervated fetuses treated with phentolamine died during the hypoxic challenge, but nine out of fourteen intact fetuses treated with phentolamine survived. 5. In intact fetuses which survived hypoxia after treatment with phentolamine, the increase in arterial blood pressure was smaller and the increase in femoral resistance did not occur. In these fetuses a rise in heart rate occurred in hypoxia. Carotid vascular resistance decreased during hypoxia after administration of phentolamine. 6. Our results indicate that the initial cardiovascular responses of the late gestation sheep fetus to hypoxia are reflex, and that the carotid chemoreceptors provide the afferent limb of this reflex. The bradycardia is mediated through a muscarinic pathway, as it is blocked by atropine. The femoral vasoconstriction is mediated through an alpha‐adrenergic mechanism, mediated both neurally by a carotid chemoreflex and via catecholamines released directly from the adrenal medulla. Both these components are blocked by phentolamine. 7. The differences in survival between intact and denervated fetuses during hypoxia after phentolamine suggest that the carotid chemoreflex response to hypoxia involves mechanisms in addition to vagal efferents to the heart and alpha‐adrenergic actions at peripheral blood vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of Altitude versus Economic Status on Birth Weight and Body Shape at Birth

The compelling evidence linking small size at birth with later cardiovascular disease has renewed and amplified a clinical and scientific interest in the determinants of fetal growth. Although the effects of maternal nutrition on fetal growth have been extensively studied, comparatively little is known about the effects of maternofetal hypoxia. This study tested the hypothesis that in highland regions, high altitude rather than maternal economic status is associated with reduced and altered fetal growth by investigating the effects of high altitude versus economic status on birth weight and body shape at birth in Bolivia. Bolivia is geographically and socioeconomically unique. It contains several highland (>3500 m above sea level) and lowland (<500 m) cities that are inhabited by very economically divergent populations. Birth weight, body length, and head circumference were compared between a high- (n = 100) and low- (n = 100) income region of La Paz (3649 m; largest high-altitude city) and a high- (n = 100) and low- (n = 100) income region of Santa Cruz (437 m; largest low-altitude city). In addition, the frequency distribution across the continuum of birth weights was plotted for babies born from high- and low-income families in La Paz and Santa Cruz. Mean birth weights were lower in babies from La Paz than in babies from Santa Cruz in both high- and low-income groups. The cumulative frequency curve across all compiled birth weights was shifted to the left in babies from La Paz compared with those from Santa Cruz, regardless of economic status. The frequency of low birth weight (<2500 g) was higher in babies from La Paz than from Santa Cruz in both high- and low-income groups. In addition, at high altitude but not at low altitude, high income was associated with an increase in the head circumference:birth weight ratio. These findings suggest that high altitude rather than economic status is associated with low birth weight and altered body shape at birth in babies from Bolivia.

Fetal cardiovascular reflex responses to hypoxaemia

The fetus mounts a coordinated cardiovascular response to an insult of acute hypoxaemia which involves neural and endocrine components. During acute hypoxaemia in late pregnancy there is a transient bradycardia, a gradual increase in arterial blood pressure and an increase in heart rate variability. In addition, there is a redistribution of the combined ventricular output favouring the cerebral, myocardial and adrenal circulations by shunting blood away from the peripheral circulations. A component of the increase in peripheral vascular resistance and the increase in arterial blood pressure during acute hypoxaemia is mediated via increases in plasma concentrations of vasoconstrictor hormones such as vasopressin, angiotensin II and neuropeptide Y. Whilst an increase in plasma ACTH and cortisol is also seen during acute hypoxaemia, their contribution to cardiovascular control in fetal sheep is less clear. Evidence has been presented to suggest that a number of these cardiovascular and endocrine responses to acute hypoxaemia are chemorefiex in nature, mediated principally by carotid chemoreceptor afferents. In addition, this reflex may be modifiable in terms of changes in magnitude and gain: first, by an influence of the intrauterine environment during chronic hypoxaemia and second, through genetic influences, in animals adapted to life at high altitude.

In vitro validation of Doppler indices using blood and water.

Using an in vitro flow model, volume flow and pressure were measured to compare calculated resistance with simultaneously measured continuous‐wave Doppler indices during imposed changes in downstream resistance to pulsatile flow. With stepwise reductions in flow, the peak (S) and trough (D) points of the maximum shifted‐frequency envelope fell in parallel in a linear fashion until D reached zero. Pressure remained constant, and the pressure waveform remained unchanged. As calculated resistance increased, resistance index (RI) and pulsatility index (PI) both increased in a linear fashion until the point at which D became zero. However, S/D ratio was not linear, and the slope increased as resistance increased. The results obtained from microspheres suspended in water and with heparinized sheeps blood were similar. The results indicate that, with reductions in flow at constant pressure in vitro, S/D ratio does not relate to calculated resistance in the same linear manner as PI and RI.

Melatonin improves placental efficiency and birth weight and increases the placental expression of antioxidant enzymes in undernourished pregnancy

Abstract:  Melatonin participates in circadian, seasonal and reproductive physiology. Melatonin also acts as a potent endogenous antioxidant by scavenging free radicals and upregulating antioxidant pathways. The placenta expresses melatonin receptors and melatonin protects against oxidative damage induced in rat placenta by ischemia‐reperfusion. One of the most common complications in pregnancy is a reduction in fetal nutrient delivery, which is known to promote oxidative stress. However, whether melatonin protects placental function and fetal development in undernourished pregnancy is unknown. Here, we investigated the effects of maternal treatment with melatonin on placental efficiency, fetal growth, birth weight and protein expression of placental oxidative stress markers in undernourished pregnancy. On day 15 of pregnancy, rats were divided into control and undernourished pregnancy (35% reduction in food intake), with and without melatonin treatment (5 μg/mL drinking water). On day 20 of gestation, fetal biometry was carried out, the placenta was weighed and subsequently analyzed by Western blot for xanthine oxidase, heat shock protein (HSP) 27 and 70, catalase, manganese superoxide dismutase (Mn‐SOD) and glutathione peroxidase 1 (GPx‐1). A separate cohort was allowed to deliver to assess effects on birth weight. Maternal undernutrition led to a fall in placental efficiency, disproportionate intrauterine growth retardation and a reduction in birth weight. Maternal treatment with melatonin in undernourished pregnancy improved placental efficiency and restored birth weight, and it increased the expression of placental Mn‐SOD and catalase. The data show that in pregnancy complicated by undernutrition, melatonin may improve placental efficiency and birth weight by upregulating placental antioxidant enzymes.

Effects of prevailing hypoxaemia, acidaemia or hypoglycaemia upon the cardiovascular, endocrine and metabolic responses to acute hypoxaemia in the ovine fetus

Although it is established that the fetus can successfully withstand a single, acute hypoxaemic challenge during gestation, little is known about what effects prevailing adverse intrauterine conditions might have on the fetal response to acute hypoxaemia. The aims of this study were therefore: (1) to characterise the effects of prevailing and sustained hypoxaemia, acidaemia or hypoglycaemia on the fetal cardiovascular responses to an episode of acute hypoxaemia; and (2) to determine the effects of these adverse intrauterine conditions on mechanisms mediating these cardiovascular responses. Thirty‐three Welsh Mountain sheep fetuses were chronically instrumented (1–2 % halothane) between 117 and 125 days of gestation (term is ca 145 days) with amniotic and vascular catheters and with a transit‐time flow probe around a femoral artery. The animals were divided retrospectively into four groups based upon post‐surgical, sustained, basal blood oxygen (chronically hypoxaemic; Pa,O2, 17.3 ± 0.5 mmHg; n= 8), glucose (chronically hypoglycaemic; blood glucose, 0.49 ± 0.03 mmol l−1; n= 6) and acid‐base (chronically acidaemic; pHa, 7.25 ± 0.01; n= 5) status. Values for compromised fetuses were −2 s.d. from a group of control (n= 14) fetuses. At 130 ± 4 days, a 1 h episode of acute, isocapnic hypoxaemia (9 % O2 in N2, to reduce carotid Pa,O2 to 12 ± 1 mmHg) was induced in all fetuses by reducing the maternal inspired O2 fraction (FI,O2). Fetal cardiovascular variables were recorded at 1 s intervals throughout the experimental protocol and arterial blood samples taken at appropriate intervals for biophysical (blood gases, glucose, lactate) and endocrine (catecholamines, vasopressin, cortisol, ACTH) measures. During acute hypoxaemia all fetuses elicited hypertension, bradycardia and femoral vasoconstriction. However, prevailing fetal compromise altered the cardiovascular and endocrine responses to a further episode of acute hypoxaemia, including: (1) enhanced pressor and femoral vasoconstriction; (2) greater increments in plasma noradrenaline and vasopressin during hypoxaemia; and (3) basal upward resetting of hypothalamic‐pituitary‐adrenal axis function. Only chronically hypoxaemic fetuses had significantly elevated basal concentrations of noradrenaline and enhanced chemoreflex function during acute hypoxaemia. These data show that prevailing adverse intrauterine conditions alter the capacity of the fetus to respond to a subsequent episode of acute hypoxaemia; however, the partial contributions of hypoxaemia, acidaemia or hypoglycaemia to mediating these responses can vary.

The role of oxygen in prenatal growth: studies in the chick embryo.

The compelling evidence linking small size at birth with later cardiovascular disease has renewed and amplified scientific and clinical interests into the determinants of fetal growth. It is accepted that genes and nutrition control fetal growth; however, prior to this study, it had been impossible to isolate the effect of increases and decreases in fetal oxygenation on the regulation of prenatal growth. We investigated the role of oxygen in the control of fetal growth in the chicken because in contrast to mammals, the effects on the fetus of changes in oxygenation could be isolated, by assessing them directly without alteration to the maternal or placental physiology or maternal nutrition during development. The data show that incubation at high altitude of fertilized eggs laid by sea level hens markedly restricted fetal growth. Incubation at high altitude of fertilized eggs laid by high altitude hens also restricted fetal growth, but to a lesser extent compared to eggs laid by sea level hens. By contrast, incubation at sea level of fertilized eggs laid by high altitude hens not only restored, but enhanced, fetal growth relative to sea level controls. Incubation at high altitude of sea level eggs with oxygen supplementation completely prevented the high altitude‐induced fetal growth restriction. Thus, fetal oxygenation, independent of maternal nutrition during development, has a predominant role in the control of fetal growth. Further, prolonged high altitude residence confers protection against the deleterious effects of hypoxia on fetal growth.

The Programming of Cardiac Hypertrophy in the Offspring by Maternal Obesity Is Associated with Hyperinsulinemia, AKT, ERK, and mTOR Activation

Human and animal studies suggest that suboptimal early nutrition during critical developmental periods impacts long-term health. For example, maternal overnutrition during pregnancy and lactation in mice programs insulin resistance, obesity, and endothelial dysfunction in the offspring. Here we investigated the effects of diet-induced maternal obesity on the offspring cardiac phenotype and explored potential underlying molecular mechanisms. Dams fed the obesogenic diet were heavier (P < 0.01) and fatter (P < 0.0001) than controls throughout pregnancy and lactation. There was no effect of maternal obesity on offspring body weight or body composition up to 8 wk of age. However, maternal obesity resulted in increased offspring cardiac mass (P < 0.05), increased heart-body weight (P < 0.01), heart weight-tibia length (P < 0.05), increased left ventricular free wall thickness and area (P < 0.01 and P < 0.05, respectively), and increased myocyte width (P < 0.001). Consistent with these structural changes, the expression of molecular markers of cardiac hypertrophy were also increased [Nppb(BNP), Myh7-Myh6(βMHC-αMHC) (both P < 0.05) and mir-133a (P < 0.01)]. Offspring were hyperinsulinemic and displayed increased insulin action through AKT (P < 0.01), ERK (P < 0.05), and mammalian target of rapamycin (P < 0.05). p38MAPK phosphorylation was also increased (P < 0.05), suggesting pathological remodeling. Increased Ncf2(p67phox) expression (P < 0.05) and impaired manganese superoxide dismutase levels (P < 0.01) suggested oxidative stress, which was consistent with an increase in levels of 4-hydroxy-2-trans-nonenal (a measure of lipid peroxidation). We propose that maternal diet-induced obesity leads to offspring cardiac hypertrophy, which is independent of offspring obesity but is associated with hyperinsulinemia-induced activation of AKT, mammalian target of rapamycin, ERK, and oxidative stress.

Reduced Cystathionine γ-Lyase and Increased miR-21 Expression Are Associated with Increased Vascular Resistance in Growth-Restricted Pregnancies: Hydrogen Sulfide as a Placental Vasodilator

Increased vascular impedance in the fetoplacental circulation is associated with fetal hypoxia and growth restriction. We sought to investigate the role of hydrogen sulfide (H2S) in regulating vasomotor tone in the fetoplacental vasculature. H2S is produced endogenously by catalytic activity of cystathionine β-synthase and cystathionine γ-lyase (CSE). Immunohistochemical analysis localized CSE to smooth muscle cells encircling arteries in stem villi. Immunoreactivity was reduced in placentas from pregnancies with severe early-onset growth-restriction and preeclampsia displaying abnormal umbilical artery Doppler waveforms compared with preeclamptic placentas with normal waveforms and controls. These findings were confirmed at the protein and mRNA levels. MicroRNA-21, which negatively regulates CSE expression, was increased in placentas with abnormal Doppler waveforms. Exposure of villus explants to hypoxia-reoxygenation significantly reduced CSE protein and mRNA and increased microRNA-21 expression. No changes were observed in cystathionine β-synthase expression, immunolocalized principally to the trophoblast, in pathologic placentas or in vitro. Finally, perfusion of normal placentas with an H2S donor, after preconstriction with a thromboxane mimetic, resulted in dose-dependent vasorelaxation. Glibenclamide and NG-nitro-l-arginine methyl ester partially blocked the effect, indicating that H2S acts through ATP-sensitive K+ channels and nitric oxide synthesis. These results demonstrate that H2S is a powerful vasodilator of the placental vasculature and that expression of CSE is reduced in placentas associated with increased vascular resistance.

Melatonin and vitamin C increase umbilical blood flow via nitric oxide-dependent mechanisms

Abstract:  Inadequate umbilical blood flow leads to intrauterine growth restriction, a major killer in perinatal medicine today. Nitric oxide (NO) is important in the maintenance of umbilical blood flow, and antioxidants increase NO bioavailability. What remains unknown is whether antioxidants can increase umbilical blood flow. Melatonin participates in circadian, seasonal, and reproductive physiology, but has also been reported to act as a potent endogenous antioxidant. We tested the hypothesis that treatment during pregnancy with melatonin increases umbilical blood flow via NO‐dependent mechanisms. This was tested in pregnant sheep by investigating in vivo the effects on continuous measurement of umbilical blood flow of melatonin before and after NO blockade with a NO clamp. These effects of melatonin were compared with those of the traditional antioxidant, vitamin C. Under anesthesia, 12 pregnant sheep and their fetuses (0.8 of gestation) were fitted with catheters and a Transonic probe around an umbilical artery, inside the fetal abdomen. Following 5 days of recovery, cardiovascular variables were recorded during fetal i.v. treatment with either melatonin (n = 6, 0.5 ± 0.1 μg/kg/min) or vitamin C (n = 6, 8.9 ± 0.4 mg/kg/min) before and after fetal NO blockade with the NO clamp. Fetal treatment with melatonin or vitamin C increased umbilical blood flow, independent of changes in fetal arterial blood pressure. Fetal NO blockade prevented the increase in umbilical blood flow induced by melatonin or vitamin C. Antioxidant treatment could be a useful clinical tool to increase or maintain umbilical blood flow in complicated pregnancy.