Michael J. Engle

Brain damage induced by prenatal exposure to dexamethasone in fetal rhesus macaques. I. Hippocampus.

Neurotoxic effects of prenatal administration of dexamethasone were examined in the fetal rhesus monkey brain at 135 and 162 days of gestation (term is 165 days). In an experimental design mimicking human clinical trials, dexamethasone was given intramuscularly to pregnant monkeys on day 132 (single injection with doses of 0.5, 5, or 10 mg/kg maternal body weight) or on days 132 and 133 (multiple injections at 12-h intervals with 0.125 x 4, 1.25 x 4, or 2.5 mg/kg x 4). The fetuses were delivered by caesarean section on day 135 or day 162 and hippocampal slices were prepared for evaluation. Light and electron microscopic observation revealed decreased numbers of pyramidal neurons in the hippocampal CA regions and of granular neurons in the dentate gyrus associated with degeneration of neuronal perikarya and dendrites. Axodendritic synaptic terminals of the mossy fibers in the CA3 hippocampal region showed pronounced degeneration. Degeneration was dose-dependent and multiple injections induced more severe damage than single injections of the same total dose. Even the lowest dose (0.5 mg/kg, which is similar to the dose used in human clinical trials) produced these changes. Degenerative changes induced by dexamethasone treatment (5 mg/kg) on days 132 and 133 were also clearly evident in fetuses studied at 162 days. Therefore, caution is recommended in the use of prenatal corticosteroids in premature deliveries.

Fatty Acid Abnormalities in Cystic Fibrosis

ABSTRACT: Fatty acids were measured by gas chromatography in lipid extracts of plasma and tissues obtained from three categories of 46 patients with cystic fibrosis. Low levels of the major essential fatty acid linoleate were found in plasma total lipids of patients who had malabsorption but not in those without evidence of steatorrhea. Circulating arachidonic acid was only slightly decreased, and the unusual triene reflecting pathologically altered fatty acid metabolism (20:3ω9) was generally not detected, nor was the triene/tetraene ratio abnormal except for in two patients. There was no correlation between plasma linoleate and age, clinical severity score, or vitamin E status. Decreased linoleate did correlate with two indices of malabsorption, namely plasma carotene (r = 0.64) and fecal fat excretion (r = 0.76). Our data therefore indicate that the abnormality in linoleate is associated with (secondary to) malabsorption of dietary fat despite pancreatic enzyme replacement therapy and consumption of a regular diet. The frequency of this alteration was determined to be quite high in 40 patients with steatorrhea, 85% of whom showed values below the lower limit of normal for plasma linoleate. It was of interest to find markedly decreased levels of linoleate in adipose tissue, cardiac muscle, and lung and lesser reductions in liver and psoas muscle taken at autopsies. Tissue arachidonic acid percentage was normal, however, and 20:3?>9 was rarely present. Thus, the physiological significance of this common abnormality in CF patients with malabsorption remains to be determined.

Hypoxic Injury to Developing Glial Cells: Protective Effect of High Glucose

ABSTRACT: Hypoxic injury to differentiating glial cells is a critical event in the development of periventricular leukomalacia, the major hypoxic-ischemic lesion of the premature infant. This study has addressed the effects of hypoxia on differentiating glial cells, primarily astrocytes. Primary cultures of dissociated newborn rat brain, which are composed predominantly of differentiating astroglia, were used. Efflux of lactate dehydrogenase, an enzyme enriched in astroglia, was used to quantitate cellular injury. Three major findings are reported. First, differentiating astrocytes were resistant to hypoxic injury for many hours, although by 24 h of hypoxia severe cellular injury (lactate dehydrogenase efflux of 86% of total and morphologic changes) was obvious. Second, increase of glucose in the culture medium from the approximately physiological concentration of 5.6 to 15 mM had a marked protective effect versus hypoxia, i.e. lactate dehydrogenase efflux was totally prevented during 24 h of hypoxia in 15 mM glucose. Third, the protective effect of high glucose appeared to be related to increased utilization by glycolysis, because there was a direct correlation between the resistance to hypoxic cellular injury and the amount of lactate generated and of glucose consumed by the cells. Thus, the cells with the lowest lactate dehydrogenase efflux (and highest glucose supplementations) had medium lactate concentrations as high as 32-36 mM. These concentrations of lactate are approximately double the reported threshold concentration of lactate considered to produce cellular necrosis in in vivo models of hypoxic injury, primarily in mature animals. The data raise the possibility that hypoxic injury to differentiating glia can be prevented or ameliorated by increase in glucose availability.

Changes in food intake during menstrual cycles and pregnancy of normal and diabetic rhesus monkeys

SummaryFood intake of control and streptozotocin-diabetic rhesus monkeys was measured during menstrual cycles and pregnancy. Intake of control monkeys was lower at the time of ovulation than during other phases of the menstrual cycle. Intake of control monkeys was also low during most of pregnancy, but this was accompanied by normal fetal growth and net maternal weight gain. Diabetic monkeys ate more than controls in all conditions and their intake did not vary reliably according to reproductive status. It is suggested that (1) oestrogen normally inhibits food intake during menstrual cycles and pregnancy, (2) food energy is utilized more efficiently during pregnancy than during non-pregnant states, and (3) the influence of oestrogen on food intake is either attenuated by insulinopenia or is obscured by the hyperphagia typically exhibited by the diabetic monkeys.

The effects of insulin and hyperglycemia on surfactant phospholipid synthesis in organotypic cultures of type II pneumocytes

Organotypic cultures of fetal type II epithelial cells were incubated in media containing insulin at concentrations ranging from 10 to 400 microunits/ml. Exposure to insulin resulted in increased glucose uptake from the media and in the rate of glucose conversion to CO2. Furthermore, both glucose uptake and CO2 production were dependent on the glucose concentration in the media. Surfactant and residual phosphatidylcholine fractions were isolated from the organotypic cultures by sucrose density centrifugation. The presence of low doses of insulin (10-25 microunits/ml) caused a significant increase in the incorporation of glucose into both surfactant and residual phosphatidylcholine. Insulin at levels of 100 microunits/ml or higher resulted in a significant decrease in glucose incorporation into both phosphatidylcholine fractions. Increasing the media glucose concentration from 5.6 to 20 mM caused a 2- to 2.5-fold increase in glucose utilization for surfactant and residual phospholipid synthesis, but did not produce any significant changes in choline incorporation into either surfactant or residual phosphatidylcholine. The addition of 400 microunits/ml of insulin to media containing 20 mM glucose, however, resulted in a 20% decrease in choline incorporation into surfactant phosphatidylcholine but had no effect on choline incorporation into residual phosphatidylcholine. These results suggest that insulin is an important hormone regulating fetal lung maturation and that hyperinsulinemia may be responsible for the delayed lung development in infants of diabetic mothers.

Fetal lung development in male and female nonhuman primates.

Abstract: Indices of lung maturation were assessed in 58 rhesus fetuses at five gestational ages during the last trimester of nonhuman primate pregnancy to determine whether fetal sex influences lung maturation. In addition to analysis of whole lung phospholipids, glycogen, protein, DNA, and pressure-volume curves surfactant fraction phosphatidylcholine (PC) was quantitated following isolation by sucrose gradient centrifugation and a combination of predictors were assessed by all possible subsets regression to attain a composite “maturity index.” For the total population, there was a uniform progression in physical growth characteristics, lung destensibility and stability and phospholipids with advancing gestation. The quantitative change in surfactant fraction PC concentration for both sexes was considerably greater than that observed for whole lung PC between 135 days gestation and term. Further, the increase in surfactant PC occurred in association with improving lung destensibility and deflation stability prior to maximum changes in the whole lung PC or disaturated PC concentration. There were no statistically discernible differences in biochemical or physiological assessment between sexes at any gestational age. These data in nonhuman primates suggest that documented differences in survival from the respiratory distress syndrome between males and females do not result from a discordance in lung maturation as a function of time throughout the last trimester of gestation.

Complications of Pregnancy and Fetal Development

Although the outcome of pregnancy for women with diabetes meilitus has improved in recent years, the infant of the diabetic mother has an increased risk of major clinical problems, particularly in the early neonatal period. These include birth injury due to macrosomia, neonatal hypoglycemia, respiratory distress syndrome, and serious congenital anomalies. Because of the great difficulties encountered during attempts to investigate these problems in clinical research protocols, there is a continuing need to establish appropriate animal models of the diabetic pregnancy. Studies carried out over the past decade, primarily with chemically-induced diabetes have suggested techniques which might be useful. In general, the choice of the animal to be studied will depend on the hypotheses being addressed. For instance, small animals such as rabbits and rats made diabetic with streptozotocin have been successfully used for investigation of fetal lung development. Furthermore, the rat model has been helpful for evaluation of fetal anomalies associated with maldevelopment of the spine and central nervous system. Larger animals, such as the nonhuman primate, are more appropriate for studying placental function and amniotic fluid composition in diabetic pregnancies. The task group on pregnancy and fetal development recommends that animal models of diabetes meilitus be used for a more extensive hormonal and metabolic characterization of diabetic mothers during pregnancy, for investigation of placental physiology with respect to the transfer of substrates from mother to fetus, for systematic and comprehensive study of mechanisms controlling fetal lung development, and for delineation of the pathophysiology of neonatal hypoglycemia. it is further recommended that animal models of spontaneous diabetes such as the BB/W rat be used in future studies dealing with pregnancy and fetal development. Because females with spontaneous diabetes show reduced conception rates, there is a pressing need to enhance the fertility of these animals in order to intensify studies on fetal development.

Changes in Surfactant Phospholipids in Fetal Rat Lungs from Normal and Diabetic Pregnancies

ABSTRACT. The purposes of this study were to adapt and evaluate further a pulmonary surfactant isolation method applicable to unperfused fetal rat lung, to quantitate key phospholipids phosphatidylcholine (disaturated phosphatidylcholine, and phosphatidylglycerol) of the isolated material during the last 3 days of gestation, and to determine if abnormalities in surfactant phospholipids were present in fetuses of diabetic pregnancies. A simplified scheme of sucrose gradient centrifugation proved useful for small scale preparations of material enriched in the phospholipids most characteristic of pulmonary surfactant. It was shown that fetal blood phospholipids did not contaminate the surfactant fraction and therefore would not produce artifacts in assessment of lung maturational changes. Analyses of subcellular fractions isolated at 19.5, 20.5, and 21.5 days revealed that the percentages of disaturated phosphatidylcholine relative to total phospholipids were 23–44% in the surfactant preparations and 14–21% in the residual (nonsurfactant) fractions, while the disaturated phosphatidylcholine/phosphatidylcholine ratios were 0.62 ± 0.06 and 0.41 ± 0.03, respectively. Summation of the amounts of individual phospholipids in the two fractions yielded data that were nearly identical to the concentrations of these compounds in whole fetal lung samples analyzed independently, implying that losses during the surfactant isolation technique were negligible. The concentrations of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, and total phospholipids increase markedly (more than 10-fold) and progressively in surfactant fractions prepared from normal fetal rat lung at 19.5, 20.5, and 21.5 days of gestation. In contrast, the residual fractions showed no changes from 19.5 to 20.5 days and then relatively modest increases from 20.5 to 21.5 days, except for phosphatidylglycerol, which increased markedly. The appearance of phosphatidylglycerol was first noted in the surfactant fraction at 20.5 days, but the level of this phospholipid did not show a marked increase until 21.5 days. These data are in agreement with previous morphologic and physiologic observations on fetal rat lung during late gestation and are also in keeping with clinical results from amniotic fluid analyses. Assessment of diabetic pregnancies revealed that at 20.5 days all phospholipids measured were significantly decreased in the surfactant fraction, but not in the residual material. Neither surfactant nor residual phospholipids were decreased in diabetic pregnancies at 19.5 or 21.5 days of gestation. The transient nature of abnormal fetal lung surfactant phospholipids in diabetic pregnancies suggests impaired timing of the pulmonary maturation processes.

Effect of dexamethasone upon surfactant phosphatidylcholine and phosphatidylglycerol synthesis in organotypic cultures of type II cells

Organotypic cultures of pulmonary type II epithelial cells were treated with dexamethasone at concentrations between 10(-10) and 10(-5) M for 48 h followed by a 3 h incubation in 5.6 mM [U-14C]glucose. A surfactant and a residual fraction was isolated from the cultures by discontinuous sucrose gradient centrifugation. Phosphatidylcholine and phosphatidylglycerol were purified from each fraction and analyzed for total content. The specific activity of each phospholipid was measured as an index of the rate of synthesis. Dexamethasone treatment produced a dose-dependent increase in synthesis and content of surfactant phosphatidylcholine, with a maximum response occurring at 10(-6) M dexamethasone. At concentrations of 10(-5) M, dexamethasone ceased to produce a significant stimulation. Dexamethasone produced an increase in surfactant phosphatidylglycerol synthesis only at a concentration of 10(-8) M and higher. There was not a significant effect upon the content or rate of synthesis of phosphatidylcholine or phosphatidylglycerol in the residual fraction at any of the dexamethasone concentrations tested.

Evidence for lactate utilization for fetal lung glycogen synthesis

Fetal rabbit lungs from 23 day gestation animals were used to investigate the potential role of lactate as a substrate for fetal lung glycogen synthesis. Fetal lactate dehydrogenase activity was approximately twice that found in the adult lung, while the activity of phosphoenolpyruvate carboxykinase was elevated fourfold over the adult value. Pyruvate carboxylase activities were similar in both fetal and adult lungs. Studies employing fetal lung explants in organ culture indicated that the presence of both glucose and lactate may be necessary for glycogen accumulation in the developing fetal lung. These data support the hypothesis that lactate is an important precursor for fetal lung glycogen.