Long-term prenatal hypoxia alters maturation of adrenal medulla in rat.

Catecholamine release from the adrenal medulla glands plays a vital role in postnatal adaptation. A number of pathologic situations are characterized by oxygen deficiency. The objective of the present study was to determine the influence of long-term prenatal hypoxia on maturation of the adrenal medulla. Pregnant rats were subjected to hypoxia (10% O2) from the fifth to the 20th d of gestation. The offspring were examined on the 19th d of gestation (E19), the day of birth (P0), and at postnatal (P) day of life P3, P7, P14, P21, and P68. The catecholamine content and activity of tyrosine hydroxylase (TH) in vivo were assayed by HPLC with electrochemical detection. Cellular expression of TH and phenylethanolamine N-methyl transferase was evaluated by protein immunohistochemistry and in situ hybridization of the corresponding mRNA species. Exposure to prenatal hypoxia reduced the epinephrine content of the adrenal medulla on E19, P0, P3, and P7 while increasing the norepinephrine content on E19, P0, and P14. Furthermore, the peak epinephrine to norepinephrine ratio appearing between P7 and P10 in the normoxic offspring was absent in the hypoxic offspring. The in vivo TH activity was increased on P3 and P14 and decreased on P68. The percentage of chromaffin cells in the medulla expressing TH and phenylethanolamine N-methyl transferase was lowered on E19, P0, and P7. TH and phenylethanolamine N-methyl transferase mRNA levels were reduced on P7. Clearly prenatal hypoxia results in major changes in adrenal catecholamine stores and synthesis during the perinatal period, which persist into adulthood. The capacity to cope with postnatal stress might be disturbed as a consequence of prenatal hypoxia.     

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??Abstract??Objective??To investigate the value of blood gas analysis in evaluating intracranial hemorrhage ??ICH?? of term neonates after hypoxia. Methods??A total of 274 full-term neonates with perinatal hypoxia or distress admitted to the First Affiliated Hospital?? Sun Yat-Sen University?? between January 2006 and January 2009?? were studied. Blood gas analysis of umbilical artery at birth and radial artery at 1 hour after birth were detected. Clinical manifestations of nervous system impairment were observed during hospital stay and cranial CT or MRI was checked within 7 days after birth to estimate ICH. Results??Totally 61 cases had ICH. ROC curve analysis showed umbilical blood pH?? BE?? umbilical and 1 hour postnatal pCO2 difference were valuable index to estimate ICH. Groups divided by umbilical or 1 hour postnatal pH?? difference of ICH incidence among groups has statistical significance ??χ2 = 17.364 and 5.314??P < 0.001 and ?? 0.023??. Difference of ICH incidence among groups divided by umbilical and 1 hour postnatal pH had statistical significance ??χ2 = 12.948?? P < 0.001??. Analysis after reject 71 forceps delivery cases?? groups divided by umbilical blood pH?? difference of ICH incidence among groups had statistical significance ??χ2 = 11.844??P = 0.003??. Groups divided by 1 hour postnatal pH?? difference of ICH incidence among groups had no statistical significance ??χ2 = 0.904??P = 0.43??. Difference of ICH incidence among groups divided by umbilical and 1 hour postnatal pH had statistical significance ??χ2 = 14.258??P = 0.003??. Conclusion??Umbilical cord combined with artery blood gas analysis after birth could be predictive of ICH in term infants after hypoxia.     

Postnatal nicotine exposure does not further compromise hypoxia defense mechanisms in prenatally nicotine-exposed lambs

AIMS: To determine whether combined pre- and postnatal nicotine exposure compared with prenatal exposure alone results in more compromised postnatal hypoxia defense mechanisms and further alteration of the postnatal breathing pattern (reduced tidal volume and increased respiratory rate). METHODS: Seven lambs exposed to nicotine prenatally (pN) (approximate maternal dose: 0.5 mg/kg/d) and seven lambs exposed to nicotine pre- and postnatally (ppN) (postnatal dose: 1.6-2 mg/kg/d) were studied without sedation at an average age of 5 d and 21 d during resting (room air) conditions, during exposure to 10% O2 and during a brief exposure to 100% O2. RESULTS: Resting minute ventilation, occlusion pressure, effective impedance, heart rate and mean arterial blood pressure were similar in the two groups during wakefulness and quiet sleep. Resting tidal volume was significantly higher in ppN than in pN lambs during wakefulness (9.4 +/- 0.7 vs 7.7 +/- 1.4 ml/kg, p < 0.05) and quiet sleep (9.8 +/- 0.6 vs 7.6 +/- 1.5 ml/kg, p < 0.01) at 5 d and also at 21 d during wakefulness (7.7 +/- 1.0 vs 6.2 +/- 1.1 ml/kg, p < 0.05). The ventilatory, heart rate and blood pressure responses to hypoxia were comparable in the two groups during both activity states. Time to arousal from quiet sleep in response to hypoxia was equivalent in the two groups. The ventilatory response to hyperoxia was not significantly different in the two groups during either activity state. CONCLUSION: Continued postnatal nicotine exposure after prenatal exposure did not further compromise hypoxia defense mechanisms after birth.     

HIF-1α特异性RNA干扰表达载体的构建及体外效应研究

目的：己知缺氧诱导因子-1α（HIF-1α）在缺氧状态下可通过调节内皮素-1（ET-1）的升高而参与肺血管内皮细胞损伤及肺动脉高压,最终导致肺出血。RNA干扰(RNAi)是双链RNA介导的序列特异性转录后同源靶基因沉默效应,目前RNAi技术已成为一种研究基因功能的有力工具,故拟通过构建人HIF-1α基因的特异性小干扰RNA（siRNA）真核表达载体,观测该载体在缺氧状态下对HIF-1α基因的干扰作用及对ET-1的抑制作用。方法：:选择6个（a～f）可能的人HIF-1α siRNA干扰位点,设计合成相应的特异性互补寡聚核苷酸链（A～F）,采用基因克隆技术,将合成的（A～F）链序列定向插入真核表达载体中,构建成重组质粒HIF-1α siRNA真核表达载体（A′～F′）,通过脂质体法转染至体外人脐静脉内皮细胞（HUVECs）48 h,再以CoCl2（100μM）模拟缺氧刺激3 h后,观测siRNA对HIF-1α mRNA和HIF-1 α蛋白表达的抑制效应及ET-1水平。结果：①成功构建了分别针对6个HIF-1 α siRNA干扰位点（a～f）的重组质粒HIF-1α siRNA真核表达载体（A′～F′）;②该6组真核表达载体转染HUVECs并经CoCl2模拟缺氧刺激后,与未转染组比较,结果显示B′ 及D′ 组明显抑制HIF-1α mRNA及HIF-1α蛋白的表达,并部分抑制ET-1表达,而其余4组与未转染组比较并无差异。结论：针对b及d干扰位点的B′及D′特异性siRNA真核表达载体,能明显干扰HIF-1α基因的表达,进而抑制ET-1的释放。该实验为下一步用B′、D′特异性siRNA真核表达载体在动物体内研究HIF-1α与新生儿缺氧性肺出血关系奠定基础。     

Effects of hypoxia on stress proteins in the piglet brain at birth

Newborn piglets were submitted to normobaric hypoxia (5% O2, 95% N2) for either 1 or 4 h. The effects of hypoxia on the neonatal brain were characterized through a time-course analysis of levels of various proteins such as heat shock proteins (HSP27, 70, and 90), hypoxia inducible factor-1alpha (HIF-1alpha), neuronal nitric oxide synthase (nNOS), hemeoxygenase-2 (HO-2), and caspase-3. The expression of these proteins was determined at different stages of recovery up to 72 h in cerebellum, cortex, and hippocampus by Western blot analysis in hypoxic maintained animals that were made hypoxic at either 20 or 37 degrees C. In all regions of the brain, HIF-1alpha and HSP27 expression were strongly increased until 22 h of recovery. No significant changes were observed for HSP70, HSP90, and HO-2. A small elevation of expression of nNOS was observed at early stages in the cerebellum and the cortex with no change in the hippocampus. Expression of caspase 3 was strongly increased in the cortex 24 and 48 h after hypoxia but unchanged in the hippocampus. These results are presented in terms of the porcine model of nonischemic hypoxia and its delayed neuronal effects on the cerebral outcome. Because of their recently established biochemical and functional interactions, the expression of the main HSPs, HIF-1alpha, nNOS, and caspase-3 after hypoxia are delineated.     

Bilateral molecular changes in a neonatal rat model of unilateral hypoxic-ischemic brain damage

Perinatal hypoxia ischemia (HI) is a frequent cause of neonatal brain injury. This study aimed at describing molecular changes during the first 48 h after exposure of the neonatal rat brain to HI. Twelve-day-old rats were subjected to unilateral carotid artery occlusion and 90 min of 8% O2, leading to neuronal damage in the ipsilateral hemisphere only. Phosphorylated-Akt levels were decreased from 0.5 to 6 h post-HI, whereas the level of phosphorylated extracellular signal-related kinases (ERK)1/2 increased during this time frame. Hypoxia-inducible factor (HIF)-1alpha protein increased with a peak at 3 h after HI. mRNA expression for IL-beta and tumor necrosis factor-alpha and -beta started to increase at 6 h with a peak at 24 h post-HI. Expression of heat shock protein 70 was increased from 12 h after HI onwards in the ipsilateral hemisphere only. Surprisingly, HI changed the expression of cytokines, HIF1-alpha ,and P-Akt to the same extent in both the ipsi- as well as the contralateral hemisphere, although neuronal damage was unilateral. Exposure of animals to hypoxia without carotid artery occlusion induced similar changes in cytokines, HIF-1alpha, and P-Akt. We conclude that during HI, hypoxia is sufficient to regulate multiple molecular mediators that may contribute, but are not sufficient, to induce long-term neuronal damage.     

Oxidative stress in preterm neonates at birth and on the seventh day of life

Previous studies have demonstrated increased oxidative damage to proteins and increased lipid peroxidation products in the plasma of hypoxic newborns at birth. We tested the hypothesis that hypoxic preterm newborns are at increased risk for oxidative stress in the first week of life. Heparinized blood samples of 34 hypoxic and 15 control preterm newborns were obtained at birth from the umbilical vein immediately after delivery and from a peripheral vein on postnatal d 7. Plasma levels of hypoxanthine, total hydroperoxide (TH), and advanced oxidation protein products (AOPP) were measured in cord blood and blood drawn on d 7. Hypoxanthine, TH, and AOPP levels were significantly higher in cord and d 7 blood samples of hypoxic newborn than control infants. Statistically significant correlations were observed between AOPP and hypoxanthine and between AOPP and TH plasma levels on d 7. AOPP and TH plasma levels significantly increased from cord to d 7 blood in neonates without hypoxia. These findings show that the oxidative stress observed in cord blood of hypoxic preterm newborns is still higher than control infants on d 7. The significant increase in TH and AOPP levels in nonhypoxic preterm newborns at the end of the first postnatal week indicates that damage caused by free radicals also occurs in nonhypoxic babies with normal clinical course. In summary, TH and AOPP production is prolonged for several days after birth in hypoxic preterm babies. The risk of free radical damage is lower but still exists in preterm neonates with normal clinical course.     

Correlative study on sex differences in pituitary luteinizing hormone content and the number of immunoreactive luteinizing hormone cells in perinatal rats

Sex differences in both pituitary luteinizing hormone (LH) content and the number of LH cells were correlatively studied in perinatal male and female rats. In the fetal pituitaries of late gestation, no sex difference was observed. On the day of birth, LH content and LH cell numbers were significantly greater in female than in male rats. Both of the two sex differences became more pronounced during the 1st and 4th postnatal days. Hormone synthesis and proliferation of pituitary LH cells are probably suppressed by testicular steroids in perinatal male rats.     

Adverse and protective influences of adenosine on the newborn and embryo: implications for preterm white matter injury and embryo protection

Few signaling molecules have the potential to influence the developing mammal as the nucleoside adenosine. Adenosine levels increase rapidly with tissue hypoxia and inflammation. Adenosine antagonists include the methylxanthines caffeine and theophylline. The receptors that transduce adenosine action are the A1, A2a, A2b, and A3 adenosine receptors (ARs). In the postnatal period, A1AR activation may contribute to white matter injury in the preterm infant by altering oligodendrocyte (OL) development. In models of perinatal brain injury, caffeine is neuroprotective against periventricular white matter injury (PWMI) and hypoxic-ischemic encephalopathy (HIE). Supporting the notion that blockade of adenosine action is of benefit in the premature infant, caffeine reduces the incidence of bronchopulmonary dysplasia and CP in clinical studies. In comparison with the adverse effects on the postnatal brain, adenosine acts via A1ARs to play an essential role in protecting the embryo from hypoxia. Embryo protective effects are blocked by caffeine, and caffeine intake during early pregnancy increases the risk of miscarriage and fetal growth retardation. Adenosine and adenosine antagonists play important modulatory roles during mammalian development. The protective and deleterious effects of adenosine depend on the time of exposure and target sites of action.     

Effects of perinatal hypoxia on serum unbound free fatty acids and lung inflammatory mediators

Cellular injury during tissue hypoxia is due, in part, to reactive intermediates released by activated leukocytes. We found that the inflammatory mediators tumor necrosis factor (TNF)-alpha, IL-6, and IL-1beta are elevated in situ in lung macrophages on day 14 following exposure of rats to intermittent or chronic hypoxia from birth. Because inflammatory mediators can increase lipolysis in adipocytes, we also measured serum unbound free fatty acids (FFAu)--the biologically active compartment of FFA--in rat pups exposed to intermittent or chronic hypoxia. FFAu values were markedly elevated during the first 2 days of life in all rats, displaying an approximately 3-fold decrease from day 2 to day 3. Exposure to chronic hypoxia significantly increased FFAu levels measured on day 13. Since elevated serum FFAu are known to suppress leukocyte activation, we speculate that increased FFAu levels represent a mechanism for attenuating inflammation and tissue injury following sublethal hypoxia in the perinatal period, either physiologically in the immediate newborn period, or as a late response to ongoing hypoxic insult.     

Impaired cardiorespiratory recovery after laryngeal stimulation in nicotine-exposed young lambs

The hypothesis that postnatal nicotine exposure weakens cardiorespiratory recovery from reflex apnea and bradycardia was tested in eight lambs continuously infused with nicotine from the day of birth at a dose of 1 to 2 mg.kg(-1).d(-1). Eight age-matched lambs infused with saline served as controls. Apnea and bradycardia were elicited by laryngeal stimulation with 1 mL of water (laryngeal chemoreflex) both during air breathing [0.21 fraction of inspired oxygen (FiO(2))] and mild hypoxia (0.10 FiO(2)) at a mean postnatal age of 5 +/- 1, 14 +/- 1, and 28 +/- 1 d. Ventilation, heart rate, and blood pressure were similar in the two groups at rest. In response to laryngeal chemoreflex stimulation, nicotine-treated lambs had a more pronounced decrease in ventilation (p < 0.05), longer reflex apnea (p < 0.001 in 0.21 FiO(2); p < 0.01 in 0.10 FiO(2)), and greater reflex bradycardia (p < 0.01). During reflex apnea, sighs were less efficient in restoring heart rate to prestimulation level, and a greater decrease in heart rate was observed before sighs in nicotine-treated lambs. These effects were most apparent at 5 d of age, when nicotine-treated lambs also had lower ventilation during hypoxia (p < 0.05). The response to hyperoxia was comparable in the two groups at all ages. The ability to terminate laryngeal chemoreflex-induced apnea is attenuated in young lambs continuously exposed to nicotine. This attenuation is present both in normoxia and in hypoxia and is accompanied by reduced effects from sighing on cardiac autoresuscitation.     

Effects of perinatal hypoxia on visual development during the first year of (corrected) age

Visual development was assessed in 124 infants (112 preterms and 12 fullterms) who had suffered from perinatal hypoxia and in 55 control preterm infants during the first year of corrected age. Using behavioural techniques, visual functions were tested during follow-up visits in the Sophia Children's Hospital. Corrected ages at testing ranged from 3 months to 1 year. During this period, infants with perinatal hypoxia showed more abnormalities in visual functions than preterm control infants. Gestational age at birth did not influence the outcome of visual development after perinatal hypoxia. Most visual impairments were demonstrated at 3 and 6 months of age. All infants with severe neuro-developmental handicaps showed visual deficits, although neuro-developmental abnormalities and visual deficits could be present as isolated phenomena. Ultrasound abnormalities related well with visual dysfunctions. Prospective studies of infants with visual deficits and a history of perinatal hypoxia are indicated.     

Expression of hypoxia-inducible factors in normal human lung development.

Pulmonary vascular development is essential for proper lung development, and its disturbance can lead to neonatal morbidity and mortality, as exemplified in congenital diaphragmatic hernia. Hypoxia-inducible factors (HIFs) appear to be key molecules in physiologic angiogenesis and in certain forms of lung pathology, such as bronchopulmonary dysplasia. Little is known about the qualitative and quantitative expression of HIFs in normal human fetal lung development. Therefore, we investigated the expression of HIF-1alpha, HIF-2alpha, and HIF-3alpha, along with their upstream regulators and downstream targets, von Hippel-Lindau protein, vascular endothelial growth factor A (VEGF-A), and its receptor, VEGFR-2, in 20 normal human fetal lungs (13.5 weeks in gestation until term) and 5 adult lungs. Quantitative polymerase chain reaction demonstrated a positive correlation between HIF-2alpha and VEGF-A expression and gestational age. Although there appeared to be a decreasing trend in HIF-3alpha expression during pregnancy, it did not reach statistical significance. Immunohistochemistry for HIF-1alpha and HIF-2alpha revealed that HIF-1alpha is expressed in the epithelium, while HIF-2alpha is expressed in both interstitium and epithelium. Our data indicate that HIFs, most notably HIF-2alpha, appear to exert an important role in angiogenesis during human fetal lung development, especially in the last phases of pregnancy, preparing the fetus for extrauterine life. As such, our results form the baseline data for the evaluation and interpretation of abnormal pulmonary vascular development.     

Free and bound tryptophan in human plasma during the perinatal period.

The concentration of tryptophan and the degree of binding of the amino acid to protein were examined in human plasma during the perinatal period. Both total and unbound (free) tryptophan were higher in cord vein plasma than in the maternal circulation, the concentration gradient being approximately 1 : 2. The proportion of the total plasma tryptophan concentration that was not bound to protein was less in cord vein plasma than in the maternal circulation. After birth the proportion in infant plasma fell significantly. Both total and free tryptophan fell during the first 24 hours of postnatal life. Total tryptophan returned to the cord vein plasma level 6--8 days after birth whilst free tryptophan failed to increase during the period of the observations. In premature infants total and free tryptophan also declined in concentration 12--24 hours after birth, suggesting the phenomenon to be related to birth rather than to gestational age. Phenylalanine remained unchanged whilst tyrosine increased in concentration during the first 80 hours of postnatal life. Thus, the availability of tryptophan to the tissues appears to decline during the immediate postnatal period and the results suggest that the requirement for tryptophan during this time may exceed the supply from standard artifical milk preparations.     

Management of the upper airway and congenital cystic lung diseases in neonates

Congenital upper airway obstruction can pose difficult problems immediately after birth. Newer strategies to maintain the airway in such situations include pharyngeal ventilation, the laryngeal mask airway and flexible fibreoptic intubation. These methods have decreased the potential for malformations such as Pierre Robin sequence to cause perinatal hypoxia. The most devastating upper airway problem is total obstruction at the supraglottic, glottic or tracheal level without tracheo-oesophageal communication. This can usually be detected prenatally and its management may include the use of the EXIT (ex-utero intrapartum) procedure. Congenital space-occupying lesions of the lung can be detected prenatally with ultrasound. Their management must be individualised, as their pre- and postnatal development is difficult to predict. Very large lesions can lead to lung hypoplasia or fetal hydrops. Management may include prenatal surgery, shunting and, rarely, an EXIT procedure during birth. A few children may require extracorporeal membrane oxygenation postnatally.