Growth restriction induced by chronic prenatal hypoxia affects breathing rhythm and its pontine catecholaminergic modulation.

Impaired transplacental supply of oxygen leads to intrauterine growth restriction, one of the most important causes of perinatal mortality and respiratory morbidity. Breathing rhythm depends on the central respiratory network modulated by catecholamines. We investigated the impact of growth restriction, using prenatal hypoxia, on respiratory frequency, on central respiratory-like rhythm, and on its catecholaminergic modulation after birth. At birth, respiratory frequency was increased and confirmed in en bloc medullary preparations, where the frequency of the fourth cervical (C4) ventral root discharge was increased, and in slice preparations containing the pre-Bötzinger complex with an increased inspiratory rhythm. The inhibition of C4 burst discharge observed in pontomedullary preparations was stronger in the growth-restricted group. These results cannot be directly linked by the tyrosine hydroxylase activity increase of A1/C1 and A2/C2 cell groups in the medulla since blockade of α1- and α2-adrenergic receptors did not abolish the difference between both groups. However, in pontomedullary preparations, the stronger inhibition of C4 burst discharge is probably supported by an increased inhibition of A5, a respiratory rhythm inhibitor pontine group of neurons displaying increased tyrosine hydroxylase activity, because blockade of α2-adrenergic receptors abolished the difference between the two groups. Taken together, these results indicate that growth restriction leads to a perturbation of the breathing frequency, which finds, at least in part, its origin in the modification of catecholaminergic modulation of the central breathing network.

Nasal OEC transplantation promotes respiratory recovery in a subchronic rat model of cervical spinal cord contusion.

Engraftment of nasal olfactory ensheathing cells (OEC) is considered as a promising therapeutic strategy for spinal cord repair and one clinical trial has already been initiated. However, while the vast majority of fundamental studies were focused on the recovery of locomotor function, the efficiency of this cellular tool for repairing respiratory motor dysfunction, which affects more than half of paraplegic/tetraplegic patients, remains unknown. Using a rat model that mimics the mechanisms encountered after a cervical contusion that induces a persistent hemi-diaphragmatic paralysis, we assessed the therapeutic efficiency of a delayed transplantation (2 weeks post-contusion) of nasal OECs within the injured spinal cord. Functional recovery was quantified with respiratory behavior tests, diaphragmatic electromyography and neuro-electrophysiological recording of the phrenic motoneurons while axogenesis was evaluated using immunohistochemistry. We show that 3 months post-transplantation, nasal OECs improve i) breathing movements, ii) activities of the ipsilateral diaphragm and corresponding phrenic nerve, and iii) axonal sprouting in the injury site. We also demonstrate that this functional partial recovery is mediated by the restoration of ipsilateral supraspinal command. Our study brings further evidence that olfactory ensheathing cells could have clinical application especially in tetraplegic patients with impaired breathing movements. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Anandamide centrally depresses the respiratory rhythm generator of neonatal mice.

Endogenous cannabinoid receptors are widely distributed throughout the CNS, including the brainstem, and modulate a variety of functions, including breathing. In adult rats, activation of cannabinoid 1 receptors has been shown to depress breathing. Here in neonatal mice, we used in vitro electrophysiology, pharmacology, and immunohistochemistry to analyse the central effects of the endocannabinoid anandamide (AEA) on the activity of the medullary respiratory rhythm generator (RRG). First of all, in vitro electrophysiology on medullary preparations has revealed that bath application of AEA (30 µM, 15 min) significantly depressed respiratory activity. Secondly, applying pre-treatments with alpha-1 (Prazosin, 5 µM, 10 min) and alpha-2 (Yohimbine, 5 µM, 10 min) adrenoceptor antagonists prior to AEA application abolished the AEA-induced depression of the RRG. Finally, immunostaining revealed a dense network of fibres positive for the cannabinoid 1 receptor in the ventrolateral medulla (VLM), a region known to contain both the RRG and the modulatory A1/C1 catecholaminergic group. Moreover, cannabinoid 1 receptor positive fibres were found in close apposition with A1/C1 catecholaminergic cells, identified by the presence of tyrosine hydroxylase. In regard of our electrophysiological, pharmacological and immunostaining results, we conclude that AEA has a central depressive effect on the neonatal RRG, probably via the medullary A1/C1 catecholaminergic neurons which are already known to modulate the respiratory rhythm generator.

Antenatal environmental stress and maturation of the breathing control, experimental data.

The nervous respiratory system undergoes postnatal maturation and yet still must be functional at birth. Any antenatal suboptimal environment could upset either its building prenatally and/or its maturation after birth. Here, we would like to briefly summarize some of the major stresses leading to clinical postnatal respiratory dysfunction that can occur during pregnancy, we then relate them to experimental models that have been developed in order to better understand the underlying mechanisms implicated in the respiratory dysfunctions observed in neonatal care units. Four sections are aimed to review our current knowledge based on experimental data. The first will deal with the metabolic factors such as oxygen and glucose, the second with consumption of psychotropic substances (nicotine, cocaine, alcohol, morphine, cannabis and caffeine), the third with psychoactive molecules commonly consumed by pregnant women within a therapeutic context and/or delivered to premature neonates in critical care units (benzodiazepine, caffeine). In the fourth section, we take into account care protocols involving extended maternal-infant separation due to isolation in incubators. The effects of this stress potentially adds to those previously described.

Developmental plasticity of the carotid chemoafferent pathway in rats that are hypoxic during the prenatal period.

The chemoreflex pathway undergoes postnatal maturation, and the perinatal environment plays a critical role in shaping respiratory control system. We investigated the role of prenatal hypoxia on the maturation of the chemoreflex neural circuits regulating ventilation in rat. Effects of hypoxia (10% O2) from the 5th to the 20th day of gestation were studied on male offspring at birth and on postnatal days 3, 7, 21 and 68. Maturation of the respiratory control system was assessed by in vivo tyrosine hydroxylase (TH) activity measurement in peripheral chemoreceptors (carotid bodies, petrosal ganglia), and in brainstem catecholaminergic cell groups (A2C2c and A1C1 areas in the medulla, A5 and A6 areas in the pons). Resting ventilation and ventilatory response to hypoxia were evaluated as functional sequelae. In peripheral structures, prenatal hypoxia reduced TH activity within the first postnatal week and enhanced it later. In contrast, in central areas, prenatal hypoxia upregulated TH activity within the first postnatal week and downregulated it later. The in vivo TH activity impairment is therefore tissue specific, with an opposite effect on the peripheral and central neural circuits. A shift of the effect of prenatal hypoxia occurred between 1 and 3 weeks, indicating a postnatal temporal effect of prenatal hypoxia. An important period in the development of the chemoafferent pathway occurred between the first and the third postnatal week. Functionally, prenatal hypoxia impaired resting ventilation and ventilatory response to hypoxia. The alterations of the catecholaminergic components of the chemoafferent pathway resulting from prenatal hypoxia might contribute to impair postnatal respiratory behaviour.

Synthesis of 2'-C-methyl-4'-thio ribonucleosides.

Starting from 2-C-methyl-ribonolactone, 1,2,3,5-tetra-O-acetyl-2-C-methyl-4-thioribofuranose was synthesized and condensed with heterocyclic bases to afford 2-C-methyl-4'-thioribonucleosides.

Synthesis of 5-aza-7-deazaguanine nucleoside derivatives as potential anti-flavivirus agents.

Coupling suitable sugars (D- or L-ribofuranose, 2' or 3-deoxysugar, branched sugars) with 2-aminoimidazo[1,2-a]-s-triazin-4-one was carried out using the different reaction conditions: 1) condensation in the presence of sodium hydride; or 2) condensation using Vorbrüggen's methods. The 5-aza- 7-deazaguanine nucleoside analogues obtained were evaluated in cell culture experiments for the inhibition of the replication of a number of RNA viruses, including BVDV, YFV, and WNV.

Neurochemical development of the brainstem catecholaminergic cell groups in rat.

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.

Increased intraabdominal adipose tissue mass in fructose fed rats: correction by metformin.

Summary. The aim of the present study was to investigate the effect of metformin on insulin sensitivity, adipose tissue mass and sympathetic nervous system (SNS) activity in fructose fed rats. Male Sprague-Dawley rats were fed for six weeks either on a standard diet (C group) or on a high-fructose diet (F group, 10% in drinking water). In each group, half of the animals received metformin in drinking water for the last 4 weeks (500 mg/kg x day, C+M and F+M). Hyperinsulinemic-euglycemic clamps (6 mU insulin/kg.min) were performed on awake unrestrained rats to test insulin resistance. Six-week fructose diet induced a reproducible insulin resistance (31.1 +/- 1.9 C vs 22.5 +/- 3.2 mg glucose/kg.min F, p<0.05). Metformin treatment prevented insulin resistance (31.1 +/- 1.9 C vs 30,2 +/- 1.8 mg glucose/kg x min F+M, ns). To measure SNS activity, rats received, ten minutes before sacrifice, an i.p. injection of NSD (m-hydroxybenzylhydrazine, inhibitor of DOPA decarboxylase, 100 mg/kg). DOPA accumulation was used as an index of SNS activity and measured in superior cervical, coeliac ganglias, retroperitoneal and epidydimal adipose tissues. SNS activity was increased in F group only in coeliac ganglia (16.8 +/- 1.1 C vs 22.6 +/- 2.2 ng DOPA/ganglia, F group, p<0.05) and not in superior cervical ganglia (8.4 +/- 0.7 C vs 8.6 +/- 0.7 ng DOPA/ganglia, F group, ns). Metformin had no effect on SNS activity in coeliac ganglia of control animals (15.9 +/- 1.7 C+M vs 16.8 +/- 1.1 ng DOPA/coeliac ganglia C, ns) but prevented the increase in SNS activity in fructose fed animals (22.6 +/- 2.2 F vs 16.3 +/- 2.8 ng DOPA/coeliac ganglia F + M). In fructose fed rats, metformin significantly increased sympathetic activity in retroperitoneal white adipose tissue (RPWAT) resulting in a marked decrease in depot mass but had no effect on epidydimal WAT. In conclusion, our results demonstrate that fructose diet caused a selective increase of SNS activity in coeliac ganglia. Metformin increased SNS activity in RPWAT resulting in a significant reduction in RPWAT mass, lowered SNS activity in coeliac ganglia to control values and restore whole body insulin sensitivity.

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.

1. 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. 2. 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. 3. 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%). 4. 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.

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.

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.