Objective
The objective of the study was to compare and contrast the effects of developmental hypoxia vs undernutrition on fetal growth, cardiovascular morphology, and function.
Study Design
On day 15 of gestation, Wistar dams were divided into control, hypoxic (10% O 2 ), or undernourished (35% reduction in food intake) pregnancy. On day 20, fetal thoraces were fixed, and the fetal heart and aorta underwent quantitative histological analysis. In a separate group, fetal aortic vascular reactivity was determined via wire myography.
Results
Both hypoxic and undernourished pregnancy was associated with asymmetric fetal growth restriction. Pregnancy complicated by hypoxia promoted fetal aortic thickening without changes in cardiac volumes when expressed as a percentage of total heart volume. In contrast, maternal undernutrition affected fetal cardiac morphology without changes in aortic structure. Fetal aortic vascular reactivity was also differentially affected by hypoxia or undernutrition.
Conclusion
Developmental hypoxia or undernutrition in late gestation has differential effects on fetal cardiovascular morphology and function.
Epidemiological and experimental evidence suggests that, in complicated pregnancy, intrauterine growth restriction (IUGR) is associated with early changes in the developing cardiovascular system. Reported abnormalities in cardiovascular morphology and function of the human IUGR fetus include an increase in relative heart weight and ventricular wall hypertrophy, a decrease in ventricle and myocyte volume, and compromised biventricular ejection force and diastolic filling. Atheromatous changes in the abdominal aorta and thickening of the aortic wall, even when adjusted for small body size, have also been documented in IUGR offspring in early childhood. In addition, studies have reported a significant inverse relationship between low birthweight and endothelial dysfunction in children in the first decade of life and in early adulthood.
In complicated pregnancy, the most common challenges to the developing fetus are reductions in nutrient and oxygen delivery. However, the partial contributions of prenatal hypoxia vs undernutrition to slow fetal growth and alterations in the structure and function of the developing cardiovascular system remain uncertain. For instance, pregnancies complicated by undernutrition result in not only a decrease in nutrient delivery to the fetus but also a decrease in placental blood flow, which will further compound nutrition as well as decrease oxygen delivery to the fetus.
Similarly, the natural hypobaric hypoxia of pregnancy at high altitude in humans and experimental maternal isobaric hypoxia in rats have both been extensively reported to promote IUGR. However, because most high-altitude populations are also impoverished and because maternal hypoxia in experimental rats can lead to a significant decrease in maternal food intake, the extent to which any effects on fetal growth and cardiovascular development is governed by fetal undernutrition or fetal underoxygenation again remains uncertain.
Using the chick embryo as an animal model, a cluster of studies have been able to isolate the effects on growth and the developing cardiovascular system of chronic hypoxia, independent of changes in maternal nutrition and of the physiology of the mother and the placenta. However, no study to date has compared and contrasted in a mammalian species the partial contributions of prenatal hypoxia vs prenatal undernutrition on alterations in fetal growth and cardiovascular development already evident prior to birth.
This study tested the hypothesis that, in mammalian complicated pregnancy, prenatal hypoxia and prenatal undernutrition have differential effects on fetal growth and on the anatomy and function of the developing cardiovascular system. The hypothesis was tested in pregnant rats by investigating the effects of maternal hypoxia vs maternal undernutrition in the last third of gestation on fetal biometry, stereological and histological changes in the fetal heart and aorta, and functional changes in fetal aortic vascular reactivity.
Materials and Methods
Animals, breeding, and housing
All procedures involving animals were carried out under the Animals (Scientific Procedures) Act of 1986. Wistar rats (Charles River Ltd, Margate, UK) were housed in individually ventilated cages (IVC units, 21% O 2 , 70-80 air changes per hour) under standard conditions (60% humidity, 21°C and a 12 hour light, 12 hour dark cycle), with free access to food (maintenance diet; Charles River Ltd) and water. After 10 days of acclimatization, virgin female Wistar rats (n = 39, 10-12 weeks of age) were paired individually with fertile male Wistar rats (minimum 12 weeks of age). The presence of a copulatory plug was considered day 0 of pregnancy (term ∼22 days). Upon the confirmation of pregnancy, the female was weighed and housed individually. Maternal weight and food and water consumption were monitored daily.
Experimental design
At day 15 of gestation, 14 pregnant females were placed inside a hypoxic chamber, which combined a PVC isolator (PFI Plastics Ltd, Keynes, UK) with a nitrogen generator (N 2 MID60, Domnick Hunter Ltd, Warwick, UK). The percentage of oxygen in the chamber was controlled by altering the inflow of air and nitrogen. Oxygen concentration was monitored continuously throughout the treatment period with an oxygen analyzer (ICA, London, UK). Pregnancies undergoing maternal hypoxia were maintained at an inspired fraction of oxygen of 10% from day 15 of gestation until day 20. The chamber was housed in the same room as the IVC units. Maternal 10% isobaric hypoxia has been shown to decrease maternal food consumption.
To discriminate between the direct effects on development of prenatal hypoxia and those induced by hypoxia-induced reductions in maternal food intake, 11 dams underwent pregnancy under normoxic conditions but were pair fed to the daily amount consumed by hypoxic dams from day 15 onward. A final group of 14 dams underwent pregnancy under normoxic conditions to serve as controls. This yielded 3 experimental groups: control (n = 14), hypoxic (n = 14), and undernourished (n = 11) pregnancy.
Tissue collection
On day 20 of gestation, some of the dams from each group (control: n = 7; hypoxia: n = 7; undernutrition: n = 5) were deeply anesthetized with ketamine (intraperitoneally, 40 mg/kg –1 , Fort Dodge Animal Health, Southampton, UK) and xylazine (intraperitoneally, 5 mg/kg –1 ; Millpledge Veterinary, Gainsborough, UK). The uterus was then exposed via a midline incision, and the anesthetized pups isolated and killed via spinal transection. In dams that had been in the hypoxic chamber from days 15 to 20 of gestation, this procedure was carried out while ventilated with 10% oxygen via a face mask. All fetuses and their associated placentae were isolated and weighed. The fetal sex was noted by measurement of the anogenital distance.
To assess the symmetry of growth, crown-rump length (CRL) was measured using digital callipers. Weights of the brain and liver were also recorded. To assess alterations in cardiovascular development, the fetal thorax from 1 male pup per litter was immersion fixed in 4% paraformaldehyde for 5 days and then processed into paraffin for subsequent stereological analysis.
The remaining dams were assigned to the in vitro wire myography study (control: n = 7; hypoxia: n = 7; undernutrition: n = 6) and killed via carbon dioxide inhalation. Fetuses were removed and body and organs weights, as well as CRL, were recorded.
Histology
Paraffin-embedded fetal thoraces, which included the heart and aorta, were exhaustively sectioned at 5 μm using a Leica RM 2235 microtome (Leica Microsystems, Heidelberg, Germany). Transverse sections were stained with Masson’s Trichrome and hematoxylin and eosin to identify cardiac muscle and smooth muscle cell (SMC) nuclei, respectively. All quantitative analyses of fixed tissue were performed using an Olympus BX-50 microscope, fitted with a motorized specimen stage and microcator (Olympus, Tokyo, Japan).
All analyses were performed using the Computer Assisted Stereology Toolbox (CAST) version 2.0 program (Olympus, Ballerup, Denmark), with the observer blind to the treatment groups. A mean value of the variable being analyzed was calculated for each animal, and a mean of means ± SEM was then obtained for each treatment group. To account for shrinkage due to paraffin processing, the diameter of erythrocytes present in sections of fetal fixed tissue was measured and compared with the diameter of erythrocytes present in fresh blood from fetal rats of the same gestational age. All measurements were subsequently corrected using this factor.
The volume of the fetal heart and its compartments was determined using a point grid, which was superimposed on the sections and viewed using a ×1.25 objective. To ensure that a precise estimate of the volumes of the different compartments of the heart and the areas of the wall and lumen of the aorta was reached, which was unaffected by inter- or intraobserver variability, approximately 200 points on 10-15 sections were counted for each animal. Points falling on the left ventricular wall plus interventricular septum, left lumen, right ventricle wall, and right lumen were counted, and the Cavalieri Principle was applied to calculate estimated volumes:
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