Adrenal phenylethanolamine-N-methyltransferase activity in the young rat: influence of thyroid hormones and insulin-induced hypoglycemia

The postnatal evolution of adrenal phenylethanolamine-N-methyltransferase (PNMT) activity is slightly accelerated by hypothyroidism and slowed by hyperthyroidism. In 14-day-old rats a single stimulation of the adrenals by insulin-induced hypoglycemia does not lead to any change in enzymatic activity. If the stimulation is repeated for 4 days, it leads to a net increase in adrenal PNMT activity in the control rats, while no increase occurs in hypo- or hyperthyroid animals.     

Effect of rat atrial natriuretic factor on in vivo and in vitro aldosterone and corticosterone secretions in the rat during the perinatal period.

The effect of rat atrial natriuretic factor (rANF) on aldosterone and corticosterone secretion was investigated in vivo in 21-day-old rat fetuses injected intravenously through the umbilical vein and in vitro on isolated adrenal cells from 17-, 19- and 21-day-old fetuses and 1-, 2-, 3- and 4-week-old rats. In vivo, rANF (50 pmol/50 microliters/fetus) inhibited both basal levels and secretion of aldosterone stimulated by adrenocorticotropin hormone (ACTH(1-24), 0.25 pmol/50 microliters/fetus), but not corticosterone secretion. In vitro, the addition of graded concentrations of rANF (0.001, 0.01 and 10 nmol/l) to the incubation medium did not affect the basal aldosterone and corticosterone secretions of fetal and neonatal adrenal cells. ACTH(1-24) (0.1 nmol/l) stimulated productions of both corticosterone and aldosterone by the adrenal cells at all stages studied. The addition of graded concentrations of rANF to the incubation medium containing ACTH(1-24) (0.1 nmol/l) induced a dose-dependent inhibition of aldosterone secretion by the adrenal cells from 21-day-old fetuses and newborn rats. In contrast, no effect was observed on cells from 17- and 19-day-old fetuses. At all stages investigated, the three doses of rANF were unable to affect ACTH-induced corticosterone secretion in vitro. In isolated adrenal cells from 2-week-old rats, rANF (10 nmol/l) inhibited the secretion of aldosterone induced by ACTH(1-24) (0.1 nmol/l), and by different steroids of the aldosterone synthetic pathway (progesterone, 11-deoxycorticosterone, corticosterone, 1 mumol/l for each steroid). These results suggest that rANF is a specific inhibitor of aldosterone synthesis in the perinatal period of the rat and that the inhibitory effect of rANF occurs both during the early and late pathways of aldosteroidogenesis.     

Differential effects of short and long durations of insulin-induced maternal hypoglycemia upon fetal rat tissue growth and glucose utilization.

We studied the effects of short and long durations of insulin-induced maternal hypoglycemia upon in vivo glucose utilization of several fetal tissues in the rat. Osmotic minipumps filled with insulin were implanted in pregnant rats on d 15 or 18 of gestation (term 21.5 d), and radiolabeled 2-deoxyglucose was used to measure relative glucose utilization rates (rGU) of fetal liver, lung, muscle, kidney, heart, placenta, and brain on d 20 of gestation after 2 or 5 d of hypoglycemia. Maternal plasma glucose concentrations decreased within 24 h of pump placement and remained less than controls throughout gestation. Fetal plasma glucose and insulin concentrations on d 20 were equally reduced after 2 and 5 d of hypoglycemia. Both 2 and 5 d of hypoglycemia were associated with significant reductions in the rGU of fetal liver, lung, and muscle. Reductions in fetal kidney rGU also occurred after 2 and 5 d of hypoglycemia but to a smaller degree. rGU of fetal heart was reduced after 2 d of hypoglycemia, but was normal after 5 d of hypoglycemia. Both 2 and 5 d of hypoglycemia were associated with increased rGU of fetal brain. Five d, but not 2 d of hypoglycemia resulted in decreased fetal weight on d 20 of gestation. However, at term, newborn pups delivered of hypoglycemic mothers weighed significantly less than controls regardless of the timing of minipump placement. Liver, lung, and carcass of these growth-retarded pups weighed less than control tissues, whereas kidney, heart, and brain weights were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neonatal sympathoadrenal response to acute hypoxia: impairment after experimental intrauterine growth retardation

Sympathoadrenal system function may be altered following intrauterine growth retardation (IUGR). We tested the hypothesis that the growth-retarded newborn rat pup has increased basal sympathoadrenal activity under normoxic conditions and a blunted sympathoadrenal response to acute hypoxia. IUGR was established by uterine artery ligation on d 18 of gestation in Sprague-Dawley rats. Growth-retarded pups were chosen as those whose birth wt was more than 2 x SD below the mean birth wt of control pups delivered to sham-operated dams. At 24 +/- 12 h of age cardiac sympathetic neuronal activity (CSNA) was determined by 3H-norepinephrine tracer and alpha-methyltyrosine techniques. Adrenal medullary catecholamine synthesis (CAT SYN) was measured by 14C-tyrosine precursor methods, and adrenal catecholamine release (CAT REL) was determined using alpha-methyl-tyrosine. IUGR and control pups were studied over a 120-min period of normoxia or hypoxia (FiO2 = 0.09). Under normoxic conditions, the IUGR pups had increased CSNA and increased adrenal CAT SYN and CAT REL compared to controls. Adrenal CAT REL in normoxic IUGR pups was selective for epinephrine. In response to acute hypoxia, control pups had increased CSNA and increased adrenal CAT SYN and CAT REL compared to normoxic controls, with the proportion of norepinephrine and epinephrine released mimicking the ratio of the two amines in the adrenal. In contrast, in hypoxic IUGR pups CSNA and adrenal CAT SYN did not increase, and norepinephrine alone was released from the adrenal medulla.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repetitive and profound insulin-induced hypoglycemia results in brain damage in newborn rats: an approach to establish an animal model of brain injury induced by neonatal hypoglycemia

The human neonate is at a higher risk for hypoglycemia-induced neuronal injury than other pediatric and adult patients. Repetitive and profound neonatal hypoglycemia can result in severe neurologic sequelae, of which the mechanisms was not elucidated by hitherto. Moreover, no reliable animal model of brain injury induced by neonatal hypoglycemia is available in order to carry out more research. Therefore, we tried to induce neonatal hypoglycemia in newborn rats by fasting and insulin injection, and then examined the neuronal degeneration after repetitive hypoglycemic insults by Fluoro-Jade B (FJB) staining. Experimental animals were randomly divided into four groups: insulin-treated rats with short hypoglycemia, insulin-treated rats with prolonged hypoglycemia, fasted rats, and control rats. Insulin injection and fasting both could induce consistent hypoglycemia in newborn rats. But from FJB staining results, only in insulin-treated rats with prolonged hypoglycemia could extensive neurodegeneration be detected. We can conclude that FJB staining is a useful method of marking neuronal degeneration in neonatal rats following hypoglycemic brain damage. Repetitive and profound neonatal hypoglycemia can result in extensive neurodegeneration, and it seems that neurons of the cortex, dentate gyrus of the hippocampus, the thalamus, and the hypothalamus are more vulnerable to hypoglycemic insult in newborn rats. Repetitive and profound insulin-induced hypoglycemia in newborn rats can establish a reliable animal model of brain injury resulting from neonatal hypoglycemia.     

Effects of cocaine on rat embryo development in vivo and in cultures

Intraperitoneal injections of 6.25, 12.5, 25, 50, and 100 mumols/kg cocaine into pregnant Sprague-Dawley rats once a day from d 0 to 19 of gestation caused a dose-dependent increase in fetal soft tissue malformations, primarily of the genitourinary tract. At 100 mumols/kg, all implants were lost and three of the five animals died after six to seven injections. At 50 and 100 mumols/kg but not at lower doses, cocaine caused a small but significant decrease in body weight and food intake. Cocaine did not affect mean fetal and placental weights, although it increased the number of runts and edematous fetuses, and did not cause skeletal malformations. After intraperitoneal injection of 50 mumols/kg, the plasma t1/2 of cocaine was 21 +/- 5 min and peak plasma concentration (1682 +/- 260 pmol/mL, measured by HPLC) was achieved in 5-10 min; a lower peak plasma concentration (486 +/- 103 pmol/mL) was achieved in 20-60 min after s.c. injection. Concentrations of dopamine, epinephrine, and norepinephrine in brains of fetuses or newborn pups (less than 12 h old) of cocaine (50 mumols/kg)-treated rats were not significantly elevated. Cocaine injections did not affect gestational duration nor the growth pattern and the locomotor activity of offspring. However, three pups born to one cocaine-treated animal died 20 d after birth. Cocaine inhibited the growth of 10.5-d-old embryos in culture in a concentration-dependent manner and was more toxic than procaine. Approximately 80% of cocaine was metabolized during a 48-h period in embryo culture medium. It is concluded that cocaine possesses teratogenic potential that may be partly independent of maternal toxicity.     

Fetal rat adrenal growth and steroidogenesis in vitro after maternal dexamethasone treatment.

Two groups of female rats were treated with dexamethasone phosphate (DEX) for 24 h. One group received DEX in drinking water (10 micrograms/ml) on the 15th day of pregnancy (DEX15--16), and the second group on the 17th day of pregnancy (DEX 17--18). The mothers and fetuses were sacrificed on the 20th and 22nd days of pregnancy. DEX reduced adrenal weights and concentration of corticosterone in plasma of the treated females and their fetuses. Fetal body weight was also reduced. However, the lightest adrenal glands were found in fetuses of the DEX17--18 mothers. Furthermore, the 19-day-old fetuses belonging to the DEX17--18 mothers had smallest amounts of corticosterone in plasma, and in plasma, and their adrenal glands produced the lowest amounts of radioactive corticosterone, 11-doxycorticosterone and 18-hydroxy-11-deoxycorticosterone from 4-14C progesterone after 1 h or incubation in vitro. In the 21-day-old fetuses belonging to both the groups of treated mothers, the production of steroids by the adrenal glands was similar and to the same extent reduced in comparison to the control fetuses.     

Significance of the 2-deoxy-d-glucose-test on the differentiation of hypoglycemia in childhood (author's transl)]

The 2-deoxy-D-glucose-test (2-DG-test) was applied to 4 children with idiopathic hypoglycemia along with some other tolerance tests. In comparison with the measurement of urinary catecholamine excretion during insulin-induced hypoglycemia, the 2-DG-test seems to be more useful because it is easier to apply and better tolerable. With regard to treatment one should differentiate between an isolated type of adrenal medullary unresponsiveness and a ketotic hypoglycemia with impaired regulation of epinephrine secretion.     

Body and organ growth in the newborn rat

Body and organ growth were described in rat pups 1 h and 10 days of age. Several organs (e.g., brain and pineal gland) exhibited significant growth in 1-hour-old but not in 10-day-old rats. Further, organs such as the testis and liver showed significant growth in 10-day-old but not 1-hour-old pups. 11 h of exposure to a warming oven, devoid of maternal and nutritional input, significantly influenced the body and organ growth of both newborn and 10-day-old pups. These data show that the first 12 h of life is an active period of growth for the rat.     

Cerebral metabolic response and mitochondrial activity following insulin-induced hypoglycemia in newborn lambs

We evaluated the newborn lamb's cerebral cellular activity and metabolism following acute insulin-induced hypoglycemia. Eleven animals received an insulin bolus followed by a continuous infusion to maintain a plasma glucose of 1 mM/l for 2 h, while 8 other animals received an equivalent dose of saline. Following the induction of hypoglycemia, the animals became quiet and transient seizures were observed in 3 animals. A significant increase in heart rate (p less than 0.01), and a decrease in arterial PaCO2 at 30 min (p less than 0.01), and pH at 2 h (p = 0.02), following hypoglycemia, were observed in the experimental group. Hypoglycemia did not significantly alter the cerebral blood flow, mitochondrial respiratory control ratio or the state-3 activity. The cerebral arteriovenous difference (CAVD) for oxygen did not change, while the glucose CAVD was significantly reduced from 0.47 +/- 0.21 to 0.24 +/- 0.16 mM/l (p less than 0.05) at the end of the hypoglycemia period, suggesting consumption of alternate substrates of energy by the brain. Insulin-induced hypoglycemia was associated with a significant increase in arterial lactate (p less than 0.01), and a significant correlation (p less than 0.01) between arterial and CAVD for lactate and beta-hydroxybutyrate (BOB) was observed. Cerebral consumption of alternate substrates of energy was inconsistent, and only observed for lactate in 5 and for BOB in 3 experimental animals following hypoglycemia. These data indicate that the newborn lamb's cerebral cellular activity is not affected by the degree of hypoglycemia achieved in these studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postnatal changes in carbamylphosphate synthetase-I and ornithine transcarbamylase activities after normal birth, premature delivery and prolonged gestation in rat liver: effects of actinomycin D and glucose.

The development of two urea cycle enzymes, carbamylphosphate synthetase-I and ornithine transcarbamylase was examined in neonatal rat liver. Normal birth on day 21.5 caused a marked increase of both activities as early as 4 h of extrauterine life. That increase occurred later in newborns delivered by cesarian section on day 21.5, but they exhibited a greater hepatic urea level. Premature delivery on day 20.5 caused a marked increase of both activities and hepatic urea level while prolonged gestation abolished these increases. The postnatal increase in both activities and in hepatic urea level was therefore dependent on a factor associated with birth. The in vivo administration of actinomycin D (2 microgram) at birth did not abolish the postnatal increase of both activities. Moreover, the administration of glucose (25 mg every 2 h) to newborns delivered by cesarian section on day 21.5 abolished the postnatal increase of ornithine transcarbamylase activity and hepatic urea level 23 h later. It seems that the transient hypoglycemia and appearance of gluconeogenesis at birth were the physiological mechanisms involved in the postnatal induction of ornithine transcarbamylase activity.     

Dynamics of glucose-induced plasma insulin increase in the rat fetus at different stages of gestation. Effects of maternal hypothermia and fetal decapitation.

The effect of glucose on the release of insulin from the pancreas of 19.5- to 21.5-day-old rat fetuses has been studied in utero. Fetal hyperglycemia was induced by a square-wave glucose infusion into pregnant rats over a period of 150 min. The infusion of glucose raised the fetal blood glucose concentration to that of the mother and induced a rapid increase of plasma insulin levels on day 19.5 of gestation. There was a progressive rise of the insulin response as the gestation proceeded, with an increase of the two phases of the hormonal secretion. Maternal hypothermia induced by pentobarbital anesthesia decreased markedly the insulin response to hyperglycemia in the mothers and their fetuses. In fetuses decapitated on day 18.5 and studied on day 21.5, the increase of plasma insulin concentration after a 1-hour hyperglycemia was similar to that in the littermate control fetuses.     

Selective ligation of uterine artery branches accelerates fetal growth in the rat

We determined if reducing litter number by selective fetal ablation (A) might enhance the growth of the surviving fetuses. On day 14 of the rat's 21.5-day gestation, we ligated branches of the uterine artery at their secondary division. Each of these branches supplies a placenta and its fetus, and ligation kills them. Every other fetus in litters of eight to 14 fetuses was ablated. Each A maternal rat was matched to a control with an equal number of fetuses. Ablation reduced fetal number (from 8-14 to 4-7) and increased fetal growth (birth weight 5.84 +/- 0.05 versus 5.20 +/- 0.05 g, p less than 0.001). In the A but not control litters, newborn body mass was negatively related to litter number. Fetuses of A litters had increased carcass, liver, and brain mass. On days 18 and 19, fetal plasma glucose concentrations, fetal/maternal glucose ratios, insulin concentrations, and hepatic glycogen concentrations were increased suggesting that A increases glucose provision to the surviving fetuses. Although these observations imply that A enhances glucose provision resulting in stimulated growth of insulin-sensitive tissues, the growth of the brain, purportedly a tissue not sensitive to insulin, was also increased. In addition, the growth rate of A fetuses continued to be increased on days 20 and 21 despite normal fetal plasma glucose and insulin. These observations indicate that with A, factors other than insulin also stimulated growth. Of note, newborn A pups developed hypoglycemia at 20 and 60 min as a result of increased insulin secretion.     

Limited maternal fuel availability due to hyperinsulinemia retards fetal growth and development in the rat

We rendered pregnant rats chronically hyperinsulinemic to determine the effect of reduced maternal metabolic fuel availability on fetal growth and development. We implanted osmotically driven insulin loaded minipumps on day 14 (term 21.5 days) in pregnant rats. This significantly increased maternal plasma concentrations of insulin and reduced glucose from day 15 until term. From day 17 until birth, fetal growth was significantly less for hyperinsulinemic mothers (term birth weight 4.53 +/- 0.07 versus 5.64 +/- 0.06 g, p less than 0.001). In fetuses of hyperinsulinemic mothers plasma glucose and insulin concentrations were significantly reduced while glucagon concentrations were increased. Total plasma amino acids were significantly reduced in maternal rats and their fetuses from days 17 to 19 while arteriovenous blood gas tensions and pH did not differ between fetuses of hyperinsulinemic and control mothers. Small for gestational age newborn pups of hyperinsulinemic mothers were hypoglycemic for the first 240 min of life as a result of limited hepatic glycogen stores and a delay in the normally expected induction of hepatic cytosolic phosphoenolpyruvate carboxykinase. This occurred despite significant increases in neonatal plasma glucagon concentrations. These data indicate that limitation of maternal glucose and amino acids with normal placental gaseous exchange retards fetal growth, limits hepatic glycogen deposition, and delays neonatal phosphoenolpyruvate carboxykinase induction. Limited fetal insulin secretion resulting from diminished maternal fuel availability may have also been a factor in retarding growth. The delay in phosphoenolpyruvate carboxykinase induction despite enhanced glucagon secretion during fetal and neonatal life suggests a specific "resistance" to this hormone in the rat growth retarded by limited metabolic fuel availability.     

Antenatal corticosteroids and newborn screening for congenital adrenal hyperplasia

OBJECTIVE: To assess the effect of reported corticosteroid exposure on neonatal levels of 17-hydroxyprogesterone (17-OHP), the cortisol precursor used in newborn screening for congenital adrenal hyperplasia, in newborns weighing less than 2500 g at birth. DESIGN: A retrospective study of newborns weighing less than 2500 g at birth and exposed to corticosteroids as reported on their newborn screening card compared with newborns weighing less than 2500 g at birth and reported as not exposed to corticosteroids. METHODS: Birth weight, gestational age, age at screening, special care information, and name of screening hospital were obtained from newborn screening cards for 16 115 newborns screened in Michigan during the first 3 months of 2000. Levels of 17-OHP, measured by fluoroimmunoassay, were obtained from Michigan's Newborn Screening Program database. RESULTS: The mean 17-OHP level for the 69 low-birth-weight newborns in the corticosteroid-exposed group was 52 ng/mL, which was higher than that for the 771 low-birth-weight newborns in the unexposed group (35 ng/mL) (P<.001). Reported corticosteroid use did not decrease the number of expected borderline positive screening results for congenital adrenal hyperplasia (P>.05). Levels of 17-OHP varied by birth weight in corticosteroid-exposed and unexposed newborns. CONCLUSIONS: Corticosteroid exposure may not suppress screening 17-OHP levels. Therefore, newborn screening should not be delayed in premature newborns because of antenatal exposure to corticosteroids.     

Hyperoxia with 100% oxygen following hypoxia-ischemia increases brain damage in newborn rats

OBJECTIVE: To describe the effect of reoxygenation with 100% O2 as compared to the effect of room air in newborn rat brains after asphyxia. METHODS: Experimental asphyxia (carotid artery ligation followed by hypoxic exposure with 8% O2 for 2 h) was performed on 7-day-old rats. After hypoxia-ischemia the rats were reoxygenated with either 100% O2 (hyperoxia group) or 21% O2 (room air group) for 24 h and then returned to the dam. The rats were killed 1 week after the experiment to study the cerebral cortex and hippocampus. RESULTS: Rats reoxygenated with 100% O2 post-asphyxia showed more frequency of cortical damage (10 of 24 rats) than those reoxy genated with room air (3 of 24 rats) (chi2 test, p = 0.02). CONCLUSION: We consider that hyperoxia with 100% oxygen after hypoxia-ischemia can cause more damage in the cerebral cortex than room air in newborn rats.     

Development of the lipolytic activity in isolated perfused perinatal rabbit lungs and the influence of maternal cigarette smoke exposure on it

The ability of the lungs to release fatty acids from circulating triglycerides or lipoproteins for its own phospholipid synthesis may be one of the factors which limit the rate of surfactant formation. Therefore, the development of the lipolytic activity of lungs obtained from late fetal and neonatal rabbits has been studied and the results correlated to the phospholipid content of lungs of similar ages. Isolated lungs were perfused with a medium which contained cold and radioactive triglyceride, and the release of fatty acids into the perfusion medium was analyzed by both colorimetric and radiochemical methods. The phospholipids of the postmitochondrial supernatant fractions of the lungs were extracted and quantified by measuring inorganic phosphorus. Finally, the influence of maternal cigarette smoke exposure on the lipolytic activity of the lungs of their litters were studied. A high lipolytic activity in the lungs of 28-day-old fetuses was detected. The activity decreased towards birth, and was lowest on the first day after birth (about 20% of that observed in 28-day-old fetuses). However, it increased again during the first week after birth. Exposure of the mothers to cigarette smoke during the last 10 days before delivery did not affect the pulmonary lipolytic activity of the offspring. Although the lung phospholipid content increased 3.6-fold from 28 days of fetal life to 1 week after birth, it remained unchanged on the days when the lung lipolytic activity was lowest. We conclude that changes in lung lipolytic activity influence lung phospholipid synthesis, and consequently influence also surfactant formation in the lungs.     

Brain excitatory amino acid concentrations are lower in the neonatal pig: a buffer against excitotoxicity?

Hypoglycemic brain damage has been associated with high levels of the excitatory amino acids (EAA) aspartate and glutamate in the newborn and adult. We hypothesized that newborn piglet EAA would be different from those of older pigs when stressed with severe insulin-induced hypoglycemia (<30 mg/dl). Brain EAA were measured in piglets and adolescent pigs via microdialysis. Eleven of 12 newborn normoglycemic piglets had no detectable baseline levels (<0.5 microM) of EAA, while pigs had aspartate and glutamate concentrations of 1.78 +/- 0.44 and 3.43 +/- 1.14 microM (mean +/- SEM), respectively. Piglet aspartate and glutamate concentrations reached but did not significantly exceed normoglycemic pig levels after 2 h with plasma glucose values < or =20 mg/ml. Elevations in EAA were only detected in piglets whose EEG activity ceased. Aspartate and glutamate concentrations did not increase in insulin-treated pigs nor in control animals. We speculate that newborns with blood glucose less than clinically acceptable values (35 mg/dl) may be protected from EAA-associated neuronal damage during acute hypoglycemia. Lower normoglycemic and hypoglycemic levels of EAA in newborns when compared to older pigs provide this protection.     

Developmental regulation of angiotensinogen gene expression in sheep

It has been suggested that the liver is not the main source of angiotensinogen during fetal life in rats, but that the kidney is an important site of fetal angiotensinogen synthesis. In an effort to determine if this phenomenon is specific to the rat or applicable to other species, we compared the ontogenic changes in hepatic and renal angiotensinogen mRNA expression in fetal (60, 90, 118, and 138 d of gestation, term being 145 d), newborn (7 d postnatal), and adult sheep. Total RNA was extracted, subjected to Northern blotting and hybridized using a full-length rat radiolabeled antisense RNA. Angiotensinogen mRNA sequences were detected in all fetal liver samples and appeared to increase 3-fold from 60 to 138 d gestation and then to decrease after birth. In contrast, angiotensinogen mRNA could not be detected in renal cortical tissue of 118 or 138 d fetuses, or newborn or adult sheep. We conclude that, unlike in the rat, liver angiotensinogen gene expression is detectable during the 2nd trimester of gestation in sheep and is developmentally regulated. Furthermore, in contrast to the fetal rat, angiotensinogen mRNA sequences were undetectable in fetal sheep kidney.