Julie Peyronnet

Long-lasting adverse effects of prenatal hypoxia on developing autonomic nervous system and cardiovascular parameters in rats

Abstract. To determine whether prenatal hypoxia increases the risk of developing cardiovascular disorders as an adult and, if so, the identity of the cell mechanisms involved in such dysfunction, we evaluated the sympathoadrenal system and central areas related to cardiovascular events during development and the cardiovascular parameters in adults. Pregnant rats were exposed to hypoxia (10% oxygen) from embryonic day (E)xa05 to E20 and the offspring studied at 1, 3, 9 and 12xa0weeks of age for neurochemistry and at 12xa0weeks of age for cardiovascular analysis. In the 1-, 3- and 9-week-old offspring, the levels and utilization of catecholamines were reduced in sympathetic ganglia, in target organs, in adrenals and in the rostral part of the A2 cell group in the nucleus tractus solitarius, but were increased in the locus coeruleus. In the 12-week-old adult offspring, the lowered autonomic nervous activity was restricted to cardiac-related structures, i.e. the stellate ganglion, heart and adrenals. In adult rats, prenatal hypoxia did not affect the cardiac parameters under resting conditions but increased blood pressure and the variability of blood pressure and heart rate under stress conditions. The altered metabolic activity of the sympathoadrenal system and related central areas during development and at adulthood for most structures might be part of the potential mechanisms contributing to cardiovascular disorders in adults.

Prenatal hypoxia impairs the postnatal development of neural and functional chemoafferent pathway in rat

1 To define the effects of prenatal hypoxia on the postnatal development of the chemoafferent pathway, ventilation and metabolism, pregnant rats were exposed to normobaric hypoxia (10 % oxygen) from embryonic day 5 to embryonic day 20. Offspring were studied at 1, 3 and 9 weeks of age in three separate protocols. 2 Prenatal hypoxia decreased the dopamine content in the carotid bodies at all ages, and decreased the utilisation rate of noradrenaline in the caudal part of the A2 (A2c), A1 and A5 noradrenergic brainstem cell groups at 3 weeks after birth. At 9 weeks of age, the level of dopamine in the carotid bodies was still reduced but the utilisation rate of noradrenaline was enhanced in A1. 3 Rats from dams subjected to hypoxia during pregnancy hyperventilated until 3 weeks after birth. In these rats, the biphasic hypoxic ventilatory response was absent at 1 week and the increase in minute ventilation was amplified at 3 weeks. 4 Prenatal hypoxia disturbed the metabolism of offspring until 3 weeks after birth. A weak or absent hypometabolism in response to hypoxia was observed in these rats in contrast to control animals. 5 Prenatal hypoxia impairs the postnatal development of the chemoafferent pathway, as well as the ventilatory and metabolic responses to hypoxia. These alterations were mostly evident until 3 weeks after birth.

Effects of gestational hypoxia on mRNA levels of Glut3 and Glut4 transporters, hypoxia inducible factor-1 and thyroid hormone receptors in developing rat brain.

Alterations of brain development result from noxious intrauterine signals, as oxygen deprivation, which decrease glucose energetic yield. To verify the hypothesis that a defect of brain energetic adaptation is responsible for these alterations, we have studied the effects of gestational hypoxia (10% oxygen during the last 2 weeks of fetal life) on cerebral ontogenesis of glucose transporters which control the limiting step of glucose utilization by neurons. This study is realised in rats by quantification of whole brain Glut3 and Glut4 mRNA in 14- and 19-day-old embryos (E14, E19), newborn (P0) and 7 postnatal-day-old rats (P7) by using reverse transcription-polymerase chain reaction (RT-PCR) method. We have associated our study with the analysis of a transcriptional factor, the hypoxia inducible factor-1alpha (HIF-1alpha), known to control the expression of glucose transporter, and with a family of transcriptional factors, the thyroid hormone receptors (TR), regulating specific genes involved in brain development. The data show (1) for the first time the Glut4 and HIF-1alpha gene expression in fetal rat brain which are detected as soon as E14, (2) that gestational hypoxia induces an increase of mRNA transcript levels of Glut3, Glut4, TRalpha2, TRbeta1 and HIF-1alpha genes mainly or exclusively at E14, and (3) that the absence of response of Glut3 and HIF-1alpha at E19 in hypoxic vs. normoxic group could indicate an insufficient energetic adaptation at this period of development which could lead to the neural alterations observed postnatally.

Ventilatory and central neurochemical reorganisation of O2 chemoreflex after carotid sinus nerve transection in rat

The first step of this study was to determine the early time course and pattern of hypoxic ventilatory response (HVR) recovery following irreversible bilateral carotid sinus nerve transection (CSNT). The second step was to find out if HVR recovery was associated with changes in the neurochemical activity of the medullary catecholaminergic cell groups involved in the O2 chemoreflex pathway. The breathing response to acute hypoxia (10% O2) was measured in awake rats 2, 6, 10, 45 and 90 days after CSNT. In a control group of sham‐operated rats, the ventilatory response to hypoxia was principally due to increased respiratory frequency. There was a large reduction in HVR in the CSNT compared to the sham‐operated rats (−65%, 2 days after surgery). Within the weeks following denervation, the CSNT rats progressively recovered a HVR level similar to the sham‐operated rats (‐37% at 6 days, −27% at 10 days, and no difference at 45 or 90 days). After recovery, the CSNT rats exhibited a higher tidal volume (+38%) than the sham‐operated rats in response to hypoxia, but not a complete recovery of respiratory frequency. Fifteen days after CSNT, in vivo tyrosine hydroxylase (TH) activity had decreased in caudal A2C2 (−35%) and A6 cells (−35%). After 90 days, the CSNT rats displayed higher TH activity than the sham‐operated animals in caudal A1C1 (+51%), caudal A2C2 (+129%), A5 (+216%) and A6 cells (+79%). It is concluded that HVR following CSNT is associated with a profound functional reorganisation of the central O2 chemoreflex pathway, including changes in ventilatory pattern and medullary catecholaminergic activity.

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.

Long-Term Impairment in the Neurochemical Activity of the Sympathoadrenal System after Neonatal Hypoxia in the Rat

The study evaluates the long-term effect of neonatal hypoxia on the neurochemical activity of the sympathoadrenal system in the rat. One-day-old male pups were exposed to hypoxia (10% O2) for 6 d and thereafter reared under normoxia. Neonatal hypoxia reduced the body weight of 3- and 8-wk-old rats and did not change the blood pressure at 6 wk of age. In sympathetic ganglia, the content and/or turnover rates of norepinephrine were reduced in neonatal-hypoxic rats of 3 and 8 wk of age, but the content and turnover rates of dopamine were unaltered. The effect was not dependent on the type of ganglion. In the superior cervical ganglion, neonatal hypoxia had a selective effect on the type of catecholamine (dopamine versus norepinephrine), thus suggesting a selective-altered maturation of noradrenergic neurons, but presumably not of the dopaminergic small, intensely fluorescent cells. A long-term deficiency in adrenal activity was the consequence of neonatal hypoxia, as shown by the decrease in the content and turnover rate of dopamine. Neonatal hypoxia elicited a long-term decrease in the content and turnover rates of norepinephrine in heart and lungs but failed to induce a significant effect in kidneys. However, this effect was not tissue-specific. Data provide evidence that a hypoxic episode occurring during a critical period of development in the rat induces a long lasting decrease in the neurochemical activity of the sympathoadrenal system. These results are discussed in terms of their implications for human pathology.

Increased Wnt levels in the neural tube impair the function of adherens junctions during neurulation.

Wnt7a has been reported to signal via the canonical pathway, but also in non-canonical pathways acting on the cytoskeleton. Since Wnt7a is expressed after neurulation, we set to investigate the effects of Wnt7a on brain regionalization. We engineered transgenic mouse embryos that, under control of the nestin second intron, overexpressed Wnt7a in neural stem/progenitor cells. Surprisingly, transgenic embryos failed to complete cranial neurulation due to reduced levels and an impaired distribution of actin microfilaments, beta-catenin, and N-cadherin at the neural tube adherens junctions. These transgenic embryos expressed high levels of Vangl2, an essential component of non-canonical Wnt signaling. In agreement with a disregulation of this pathway, aberrant spinal neurulation was detected in the transgenic embryos, revealing a novel function regulated by Wnts. Thus, our findings suggest that Wnt7a overexpression disrupts normal Wnt signaling in the neural tube, resulting in defective adherens junctions and neurulation.

Neurochemical development of the brainstem catecholaminergic cell groups in rat.

Summary. The postnatal development of tyrosine hydroxylase activity has been studied in the brainstem catecholaminergic cell groups (A1C1, A2C2, A5, A6, A7), involved in cardiorespiratory control. In rat, at birth and at postnatal days P3, P7, P14, P21 ant P68, we used a microdissection technique followed by in vivo measurement of the tyrosine hydroxylase (TH) activity, the rate-limiting enzyme in catecholamine synthesis. There is two successive marked increases in TH activity: at P3 in every catecholaminergic cell groups (A1C1, +225%; A2C2, +300%; A5, +190%; A6, +205% compared to birth) and during the third postnatal week with a peak of TH activity at P14 (A6, +90% above the P7 level) or at P21 (A1C1, +715%; caudal A2C2, +585%; rostral A2C2, +15%; A5, +445%; A7, +180% compared to P7). The data suggest the existence of two temporal windows during the neurochemical development of the catecholaminergic cell groups, which correspond to two metabolic transitions. The first one could be related to the intra-, extrauterine transition and the second one, to a deep energetic phase of maturation in the rat brain, closely related to the maturation of cardiorespiratory processes.

Plasticity in the phenotypic expression of catecholamines and vasoactive intestinal peptide in adult rat superior cervical and stellate ganglia after long-term hypoxia in vivo

Sympathetic ganglia in the adult rat contain various populations of nerve cells which demonstrate plasticity with respect to their transmitter phenotype. The plasticity of the neuronal cell bodies and of the small intensely fluorescent cells in the superior cervical and stellate ganglia in response to hypoxia in vivo (10% O2 for seven days) was assessed by studying the expression of catecholamines and vasoactive intestinal peptide. The levels of norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid and vasoactive intestinal peptide immunoreactivity were determined. In addition, the density of the immunohistochemical staining of cells for tyrosine hydroxylase and vasoactive intestinal peptide was evaluated. In the intact superior cervical ganglion, hypoxia increased the dopamine level as well as the density of small intensely fluorescent cells immunolabelled for tyrosine hydroxylase and vasoactive intestinal peptide. In the axotomized ganglion, hypoxia elicited a twofold rise in the level of the vasoactive intestinal peptide as well as enhancing the density of neuronal cell bodies immunostained for this peptide. Thus, the effect of hypoxia on the expression of vasoactive intestinal peptide expression in neurons was dependent on neural interactions. In the intact stellate ganglion, hypoxia alone induced a 1.5-fold increase in the density of neuronal cell bodies immunostained for vasoactive intestinal peptide. Thus, ganglia-specific factors appeared to play a role in determining changes in neuronal phenotype in response to hypoxia. The present study provides evidence for the involvement of dopamine and vasoactive intestinal peptide in ganglionic responses to long-term hypoxia as well as for differential responses by the two ganglionic cell populations, i.e. neuronal cell bodies and small intensely fluorescent cells. Changes in the expression of the vasoactive intestinal peptide during long-term hypoxia may be of energetic, trophic and/or synaptic significance. Hypoxia may be considered to be a vasoactive intestinal peptide-inducing factor in sympathetic ganglia.

Prenatal nicotine alters maturation of breathing and neural circuits regulating respiratory control.

While perinatal nicotine effects on ventilation have been widely investigated, the prenatal impact of nicotine treatment during gestation on both breathing and neural circuits involved in respiratory control remains unknown. We examined the effects of nicotine, from embryonic day 5 (E5) to E20, on baseline ventilation, the two hypoxic ventilatory response components and in vivo tyrosine hydroxylase (TH) activity in carotid bodies and brainstem areas, assessed at postnatal day 7 (P7), P11 and P21. In pups prenatally exposed to nicotine, baseline ventilation and hypoxic ventilatory response were increased at P7 (+48%) and P11 (+46%), with increased tidal volume (p<0.05). Hypoxia blunted frequency response at P7 and revealed unstable ventilation at P11. In carotid bodies, TH activity increased by 20% at P7 and decreased by 48% at P11 (p<0.05). In most brainstem areas it was reduced by 20-33% until P11. Changes were resolved by P21. Prenatal nicotine led to postnatal ventilatory sequelae, partly resulting from impaired maturation of peripheral chemoreceptors and brainstem integrative sites.