OBJECTIVE: Inhaled nitric oxide (iNO) improves oxygenation and reduces the need for extracorporeal membrane oxygenation in infants with severe persistent pulmonary hypertension of the newborn (PPHN). The effectiveness of iNO in the treatment of moderate PPHN has not been adequately defined. We therefore conducted a randomized, prospective multicenter study to assess whether iNO in patients with moderate PPHN would improve arterial paO2, prevent progression to severe PPHN, and improve outcomes. METHODS: Infants ≥34 weeks gestation with moderate pulmonary hypertension (alveolar–arterial oxygen gradient (AaDO2) 500–599 Torr) were randomly assigned to continue standard medical therapy (control group) or standard medical therapy plus iNO (iNO group). For each patient in the iNO group, iNO concentration was increased in steps of 10–20 ppm every 30 minutes until there was no further improvement in arterial paO2. This concentration of iNO was then maintained while all other ventilatory support, including inspired oxygen concentration, was weaned according to a predefined protocol. RESULTS: In all, 27 of 40 control patients (58%) compared to six of 40 infants (15%) in the iNO group failed assigned therapy and developed severe PPHN (p<0.0005). Arterial paO2 improved from 112±48 to 133±100 (p=0.132) in control infants compared to an increase from 101±29 to 208±118 (p<0.0005) in iNO-treated patients. For the first 36 hours after study, entry AaDO2 levels and ventilatory support were significantly lower in iNO-treated infants compared to control patients. CONCLUSION: In patients with moderate PPHN, treatment with iNO improves arterial paO2, reduces the amount of ventilatory support needed, and prevents progression to severe PPHN.

Using hyt/hyt mice that exhibit naturally occurring primary hypothyroidism (n = 72) and Balb/c controls (n = 66), we examined the mRNA, protein, and activity of brain glucose transporters (Glut 1 and Glut 3) and hexokinase I enzyme at various postnatal ages (d 1, 7, 14, 21, 35, and 60). The hyt/hyt mice showed an age-dependent decline in body weight (p < 0.04) and an increase in serum TSH levels (p < 0.001) at all ages. An age-dependent translational/posttranslational 40% decline in Glut 1 (p = 0.02) with no change in Glut 3 levels was observed. These changes were predominant during the immediate neonatal period (d 1). A posttranslational 70% increase in hexokinase enzyme activity was noted at d 1 alone (p < 0.05) with no concomitant change in brain 2-deoxy-glucose uptake. This was despite a decline in the hyt/hyt glucose production rate. We conclude that primary hypothyroidism causes a decline in brain Glut 1 associated with no change in Glut 3 levels and a compensatory increase in hexokinase enzyme activity. These changes are pronounced only during the immediate neonatal period and disappear in the postweaned stages of development. These hypothyroid-induced compensatory changes in gene products mediating glucose transport and phosphorylation ensure an adequate supply of glucose to the developing brain during transition from fetal to neonatal life.

Diverse Effects of Intrauterine Growth Retardation (IUGR) upon Mitchondrial Gene Expression and Function in Male and Female d120 Soleus and Extensor Digitalis Longus (EDL) Muscle † 448

The epidermal permeability barrier, required for terrestrial life, is localized to lipid-enriched lamellar membranes in the extracellular spaces of the stratum corneum (SC). Immaturity of the SC is a significant contributor to morbidity and mortality in premature infants. Previous studies have shown that supraphysiologic concentrations of thyroid hormone accelerate epidermis/SC ontogenesis. Here we studied SC development in Hyt/Hyt mice who are genetically hypothyroid due to a mutation in the TSH receptor. In control mice on d 18 of gestation (term 19.5 d), only focal areas displayed a mature SC membrane pattern. By 19 d of gestation there was a mature multilayered SC with lamellar unit structures filling the extracellular spaces similar to that seen in mature mice. In Hyt/Hyt mice SC development was delayed at both 18 and 19 d of gestation. In both strains of mice, within the first day after birth there were no differences in epidermal or SC appearance, and the SC was fully mature. These findings indicate that thyroid hormone plays a physiologic role during normal intrauterine development of the SC. However, normal SC maturation ultimately occurs, indicating that thyroid hormone is not absolutely essential. Previous studies have shown that glucocorticoids accelerate SC development in euthyroid rats, and in the present study we demonstrate that glucocorticoids also accelerate SC ontogenesis in euthyroid mice. In contrast, in Hyt/Hyt mice glucocorticoids did not accelerate or normalize SC development, indicating that the glucocorticoid effect on SC maturation requires a euthyroid state or that glucocorticoids act via thyroid hormone. These studies demonstrate that thyroid hormone status is an important regulator of fetal SC development.

Maternal administration of TSH-releasing hormone (TRH) in the euthyroid mouse accelerates fetal lung ultrastructural maturation. However, the mechanism(s) of TRH in fetal lung development remains unclear; it could be due to its neuroendocrine and/or neurotransmitter effects. Although the neuroendocrine effect of TRH is mediated via stimulation of the fetal pituitarythyroid axis, the neurotransmitter effect is mediated via stimulation of fetal autonomic nervous system activity. In the hyt/hyt mouse there is a point mutation in the β subunit of the TSH receptor in the thyroid gland of the Balb-c mouse. In these mice TSH does not bind to its receptors, leading ultimately to the development of primary hypothyroidism, which is transmitted as an autosomal recessive trait. A maturational delay in the lung ultrastructure of the hyt/hyt mouse fetus has been observed. This investigation was undertaken to study the effect of maternal TRH treatment on lung ultrastructural maturation in the hyt/hyt mouse fetus. If the effect of TRH is mediated via stimulation of fetal pituitary-thyroid axis, TRH treatment should not enhance lung maturity in the hyt/hyt fetus and vice versa. Adult hyt/hyt mice made euthyroid by triiodothyronine supplementation were mated to carry hyt/hyt pups. Saline or TRH (0.4 or 0.6 mg/kg/dose) was administered to the mother (i.p.) on d 16 and 17 (b.i.d.) and on d 18 pregnancy 1 h before killing (term, ≈20 d). The fetal lung electron micrographs were subjected to ultrastructural morphometric analysis of the number of lamellar bodies and glycogen/nuclear ratio in type II cells, and the alveolar/parenchymal ratio by Chalkley point counting with an interactive computerized image analyzer (Optimas, Bioscan). Fetal lungs exposed to the lower dose of TRH (n = 7) showed no significant difference in their ultrastructural maturation when compared with saline-treated controls (n = 5). However, fetal lungs exposed to a higher dose of TRH (n = 6) showed increased numbers of lamellar bodies per type II cell, an increase in the alveolar/parenchymal ratio, larger air spaces, thinner alveolar septa, presence of tubular myelin, and increased numbers of air-blood barriers. We conclude that the effect of TRH in accelerating fetal mouse lung maturation is at least in part mediated via stimulation of extra thyroidal pathways.

Primary hypothyroidism in the Hyt/Hyt mice is due to a point mutation(Pro-556-Leu) in the β subunit of the TSH receptor of the thyroid gland and is transmitted as an autosomal recessive trait. Fetal hypothyroidism is associated with delayed fetal lung ultrastructural maturation (Ped. Res. 36:380:94). Surfactant proteins (SP-A, B & C) & Thyroid Transcription Factor-1 (TTF-1) play an important role in fetal lung surfactant function and type II cell differentiation, but the effect of primary hypothyroidism on the developmental expression of fetal lung SP-A, SP-B, SP-C or TTF-1 has not been investigated. Hyt/Hyt mice identified by high serum TSH and low free T4 levels were made euthyroid by supplemental T3. The mice were then bred to carry Hyt/Hyt pups. Balb-c euthyroid mice served as controls. All mice were killed on d 18 of gestation (term ≈20 d, vaginal plug = d 1 of pregnancy). Fetal lung immunostaining for SP-A, SP-B, SP-C and TTF-1 was performed and gene expression of these proteins was determined by Northern blot analysis. The TTF-1 immunoreactivity was nuclear in localization and confined to the type II cells, while staining for SP-A, SP-B and SP-C was cytoplasmic and present in type II cells as well as within the alveolar lumen. The intensity and distribution of the immunoreactivity scored on a scale of 0 to 3, for SP-A, SP-B and SP-C and TTF-1 (n=5) was markedly decreased in the Hyt/Hyt fetal lungs when compared to the controls (n=5). While gene expression for SP-C(≈0.8 kb) and TTF-1 (≈2.3 kb) was significantly decreased in the hyt/hyt mice, the gene expression for SP-B (≈0.9 kb) was similar in both groups. CON: Primary hypothyroidism is associated with a delay in the developmental expression of fetal lung SP-A, SP-B, SP-C and TTF-1 protein, and a differential expression for SP-B, SP-C and TTF-1 genes. These findings may explain a higher incidence of RDS in human neonates with hypothyroidism(NIH-HL 52839).

Glucose, an essential substrate for brain growth, cellular maturation, and oxidative metabolism is transported across the blood-brain barrier, into neurons and glial cells. Intracellularly glucose is phosphorylated into glucose-6-phosphate by the hexokinase I (hxl) enzyme. In the adult brain, glucose phosphorylation comprises the rate-limiting step in the process of glucose uptake. To determine the ontogeny of this critical rate limiting step, we examined the brain hxl expression (Northern blots), concentration (Western blots), enzymatic activity (NADPNADPH conversion by spectrophotometric assay) and function (3H-2-deoxy-glucose uptake) in Balb-C mice at 1d(n=6), 14d (n=6), and 35d (n=6) postnatal ages. Hxl mRNA and protein concentrations declined 20-50% (p < 0.05) while activity increased 2-fold between the 1d and 14d or 35d brains. In contrast, a six-fold increase in3 H-2-deoxy-glucose uptake (p < 0.05) which quantitates glucose transport and phosphorylation was noted between the 1d and 14d old mice. Our previous investigation demonstrated a 3-fold increase in brain glucose transporter expression and levels (particularly neuronal Glut 3) between the 1d and 14d mouse brains (Ped Res 39:91A, 1996). We conclude that 1] brain Hxl enzyme activity and function peak by a post-translational mechanism at the 14d postnatal age, 2] the age-related increase in brain Hxl enzyme activity along with the previous observation of a parallel increase in brain glucose transporter concentrations substantiate the age-related increase in brain 2-deoxyglucose uptake with a peak at 14d postnatal age. We speculate that this age-dependent increase in the mechanisms mediating brain glucose uptake 1] may be initiated by the physiological surge in thyroid hormone levels and activity that occurs at 14d of age, and 2] is critical for fueling the process of rapid brain growth and cellular development that occurs at 14d postnatal age in preparation for acquiring the specialized function of neurotransmission.

Fetal Thyroid Plays an Important Role in Acceleration of Fetal Mouse Lung Ultrastructural Maturation from Glucocorticoid Stimulation. † 1454

Primary hypothyroidism alters expression and function of the hexokinase I gene which mediates brain glucose phosphorylation. † 1390

Effect of Maternal Betamethasone, Thyrotropin Releasing Hormone and Betamethasone plus Thyrotropin Releasing Hormone Therapy on Mouse Fetal Lung Surfactant Protein A, B and C Expression. † 1455

Thyroid hormones play an important role in mammalian fetal lung development. Delayed fetal lung ultrastructural maturation is observed in the Hyt/Hyt mouse which has primary hypothyroidism due to a point mutation(Pro-556-Leu) in the β subunit of the TSH receptor (Ped. Res.380-386,1994). Thyroid transcriptional factor 1 (T.T.F.-1) and surfactant protein C (SP-C) play an important role in fetal lung function and type II cell differentiation. The effect of primary hypothyroidism on the developmental expression of lung SP-C or TTF-1 has not been studied. Hypothyroid (Hyt/Hyt) mice, characterized by high serum TSH and low serum free T4 concentration, were made euthyroid by T3 supplementation. These mice were mated to produce hypothyroid (Hyt/hyt) pups. Balb-c mice served as euthyroid controls. Mice were killed on d 18 of pregnancy (term≈19d, vaginal plug=d 1). Immunostaining of fetal lung for SP-C was done using polyclonal antibody. The intensity of the immunoreactivity for SP-C was scored on a scale of 0 to 3 in 0.5 increments. Fetal lung gene expression for T.T.F.-1 and SP-C was determined by Northern analysis and densitometry. All data Mean ±SEM, * P<0.02 There was a linear relationship between the gene expression for T.T.F.-1 and SP-C in these mice (r=0.86). We conclude that primary hypothyroidism causes a delay in the gene expression for TTF-1 and SP-C in the fetal lung. Table

Effects of early onset bacterial sepsis (EOBS) and pregnancy induced hypertension (PIH) on the first white blood cell (WBC) and platelet counts done within the first 8 hours of life in infants less than 1,200 g and 32 weeks of gestation (n = 121) were analyzed. PIH (n = 24) but not EOBS (n = 19) was associated with leukopenia while both PIH and EOBS were accompanied by absolute neutropenia. PIH and EOBS influenced immature to total neutrophil ratios. However, immature to total neutrophil ratio was not a good predictor of EOBS in these very low birth weight (VLBW) infants. PIH but not EOBS was associated with thrombocytopenia. We conclude that the first WBC and platelet counts are not very useful in predicting EOBS in VLBW infants, and that the factor(s) causing abnormal neonatal WBC and platelet counts in PIH pregnancies are operational much earlier in pregnancy than previously recognized.

Plasma concentrations of 25-hydroxy- and 1,25-dihydroxyvitamin D in chronically catheterized fetal and maternal sheep preparations and transfer of ^3H-1,25- (OH)2-D(3) from the fetus to mother and vice versa were studied. We tested the hypothesis that l,25-(OH)(2)-D does not cross the placenta in either direction. While the fetal plasma 25-(OH)-D concentration was lower than the corresponding maternal values (n = 9, p < 0.02), the fetal 1,25-(OH)(2)-D concentration was higher than the mother’s (n = 9, p < 0.02). 3H-1,25-(OH)(2)-D(3) crossed the placenta from the fetus to the mother (n = 4) and vice versa (n = 4). We speculate, therefore, that maternal vitamin D metabolism may be affected by fetal vitamin D metabolism through the transplacental crossover of its highly active metabolite.