Glial vascular endothelial growth factor overexpression in rat brainstem under tolerable hypoxia: evidence for a central chemosensitivity.

The ventilatory response to hypoxia is mediated by peripheral inputs arising from the arterial chemoreceptors. In their absence, hypoxic adaptation can be achieved, possibly as a result of central cellular reorganization. To study this reorganization, we used chemodenervated rats to investigate the expression and localization of vascular endothelial growth factor (VEGF) in the brainstem. VEGF is a target gene of hypoxia-inducible factor (HIF) that is responsible for the morphofunctional remodeling induced by hypoxia. Intact and chemodenervated rats were subjected to normoxia or hypoxia for 6 hr (10% O(2) in N(2)). VEGF protein was quantified in micropunches of brainstem tissue. Only chemodenervated animals showed an increased VEGF expression in response to hypoxia, whereas, in normoxia, VEGF expression was not modified by chemodenervation. The same hypoxic condition was repeated for 8 days before immunocytochemical staining with anti-VEGF; antiglial fibrillary acidic protein (GFAP), a marker of astrocytes; and anti-rat endothelial cell antigen-1 (anti-RECA-1) that recognizes endothelial cells. Confocal analysis showed a cellular colocalization of GFAP and VEGF, indicating that VEGF was overexpressed predominantly in astrocytes. Increased RECA-1 immunolabeling indicated an enhanced angiogenesis in chemodenervated rats subjected to hypoxia. These results indicate that glial cells and the vascular network contribute to the brainstem remodeling. The peripheral chemodenervation reveals a central O(2) chemosensitivity involving a cascade of gene expression triggered by hypoxia, which in intact animals may act synergically with peripheral chemosensory inputs.

Ret deficiency in mice impairs the development of A5 and A6 neurons and the functional maturation of the respiratory rhythm.

Although a normal respiratory rhythm is vital at birth, little is known about the genetic factors controlling the prenatal maturation of the respiratory network in mammals. In Phox2a mutant mice, which do not express A6 neurons, we previously hypothesized that the release of endogenous norepinephrine by A6 neurons is required for a normal respiratory rhythm to occur at birth. Here we investigated the role of the Ret gene, which encodes a transmembrane tyrosine kinase receptor, in the maturation of norepinephrine and respiratory systems. As Ret-null mutants (Ret-/-) did not survive after birth, our experiments were performed in wild-type (wt) and Ret-/- fetuses exteriorized from pregnant heterozygous mice at gestational day 18. First, in wt fetuses, quantitative in situ hybridization revealed high levels of Ret transcripts in the pontine A5 and A6 areas. Second, in Ret-/- fetuses, high-pressure liquid chromatography showed significantly reduced norepinephrine contents in the pons but not the medulla. Third, tyrosine hydroxylase immunocytochemistry revealed a significantly reduced number of pontine A5 and A6 neurons but not medullary norepinephrine neurons in Ret-/- fetuses. Finally, electrophysiological and pharmacological experiments performed on brainstem 'en bloc' preparations demonstrated impaired resting respiratory activity and abnormal responses to central hypoxia and norepinephrine application in Ret-/- fetuses. To conclude, our results show that Ret gene contributes to the prenatal maturation of A6 and A5 neurons and respiratory system. They support the hypothesis that the normal maturation of the respiratory network requires afferent activity corresponding to the A6 excitatory and A5 inhibitory input balance.

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.

Altered daily rhythms of brain and pituitary indolamines and neuropeptides in long-term hypoxic rats.

To determine whether sustained hypoxia alters daily rhythms in brain and pituitary neurotransmitters, the daily variations in vasoactive intestinal peptide-like immunoreactivity (VIP-LI), neuropeptide Y-like immunoreactivity (NPY-LI), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) content were determined in discrete brain regions, pineal gland and anterior pituitary of hypoxic (10% O(2); 14 days) and normoxic rats. Hypoxia suppressed daily variations in VIP-LI in the suprachiasmatic nuclei (SCN) and the anterior pituitary, enhanced the daily rhythmicity in serotonergic elements of the caudal part of the dorsomedial medulla oblongata (DMMc), and even induced daily variations in NPY-LI in the DMMc as well as in the ventrolateral medulla oblongata. In addition, punctual alterations in the rhythmicity of 5-HT and 5-HIAA in the pineal gland and of plasma corticosterone were observed in hypoxic rats. Thus results of this study indicate that a permanent nonphotic stimulus, such as sustained hypoxia, may affect the functioning of the internal clock located in the SCN and may alter the daily rhythmicity in neurotransmitter content of some brain nuclei and the pituitary gland.

Differential effects of ventilatory stimulation by sex hormones and almitrine on hypoxic erythrocytosis.

In the absence of pulmonary disease, hypoventilation is considered to be the primary cause of Chronic Mountain Sickness, and there is some reason to believe that chronic administration of respiratory analeptics could be useful for treatment of this disease. The present study was intended to define comparatively the influence of two potent ventilatory stimulants, namely a combination of progesterone and estrogen and the pharmacological agent almitrine, on catecholaminergic structures implicated in the chemoreflex pathway and on hypoxia-induced polycythemia. Three groups of young male rats born and living at high altitude (3 600 m) were examined: untreated animals (n = 25), rats given ovarian steroids (progesterone plus 17beta-estradiol, n = 25) or almitrine (n = 25) for 6 weeks until sacrifice. Ovarian steroids or almitrine had pronounced neurochemical effects on the afferent chemoreflex circuitry. Both treatments inhibited norepinephrine (NE) and dopamine (DA) turnover in the carotid body, but central processing of chemosensory inputs differed between the two respiratory drugs. Ovarian steroids inhibited noradrenergic activity in the projection area of the chemosensory nerve fibers within the caudal portion (A2C) of the nucleus tractus solitarius (NTS). In contrast, almitrine stimulated neurochemical activity of other brainstem noradrenergic cell groups involved in cardiorespiratory control, i.e. , the rostral portion (A2R) of the NTS, the nucleus reticularis lateralis (A1), the nucleus olivaris superior (A5) and the locus ceruleus (A6). Although both treatments increased chemoreflex drive and ventilation, only sex hormones decreased erythropoietin (EPO) levels and the degree of polycythemia. These results suggest that stimulation of ventilation through activation of peripheral arterial chemoreceptors activation alone is not sufficient for reducing EPO levels and polycythemia. The better efficiency of female sex hormone treatment as compared to almitrine could be related either to the central effects of progesterone and estrogen and/or to the impact of these hormones on erythropoiesis at the kidney/bone marrow level.

Alteration in central and peripheral substance P- and neuropeptide Y-like immunoreactivity after chronic hypoxia in the rat.

The influence of long-term hypoxia on substance P (SP) and neuropeptide Y (NPY)-like immunoreactivity (LI) in discrete brain areas and peripheral structures was assessed by radioimmunoassay. Rats were exposed to normobaric hypoxia (10% O2 in nitrogen) for 14 days. In the carotid bodies of hypoxic animals, NPY-LI was significantly increased (56% vs. normoxic controls) while SP-LI was unchanged. In the brain, NPY-LI was increased in the ventrolateral medulla oblongata (23%) and in the striatum (53%); however, SP-LI was unaltered in these two regions. In the anterior pituitary, NPY-LI was increased (99%), while SP-LI was decreased (37%). No significant alteration in NPY-LI and SP-LI was observed in other discrete brain areas or peripheral structures studied. These results show that, in the rat, long-term hypoxia induces changes in NPY-LI or SP-LI in a few central and peripheral structures; these biochemical alterations may be linked to adaptative mechanisms involving morphological changes in carotid bodies or alterations in sympathetic control and neuroendocrine function.

Chronic hypoxia affects peripheral and central vasoactive intestinal peptide-like immunoreactivity in the rat.

The influence of long-term hypoxia on vasoactive intestinal peptide-like immunoreactivity (VIP-LI) in discrete brain areas and peripheral structures was assessed by RIA. Rats were exposed to normobaric hypoxia (10% O2-90% N2) for 14 days. VIP-LI was significantly increased in carotid bodies of hypoxic animals (204% vs. normoxic animals). On the other hand, VIP-LI was decreased in the anterior pituitary (-68%), suprachiasmatic nuclei (-29%) and periventricular nuclei (-26%). No significant variation in VIP-LI was observed in other peripheral structures and discrete brain area studied. These results suggest that long-term hypoxia induces alterations in VIP systems implicated in chemoreception, biological rhythms and neuroendocrine functions.

Effect of almitrine administration on pulmonary arterial pressure in resting and exercising dogs.

In addition to its well-known ventilatory effect, a small rise in pulmonary arterial pressure or pulmonary vascular resistance is occasionally observed with chronic administration of almitrine. In order to test the hypothesis of enhancement of exercise pulmonary vasoconstriction by almitrine, mongrel dogs were studied at rest and during submaximal exercise before and after 4 weeks of chronic ingestion of almitrine (10 mg/kg). It was shown that resting pulmonary arterial pressure (PAP) remained unchanged by almitrine treatment. However, when exercise was superimposed on almitrine medication, PAP was significantly increased throughout the exercise bout. Thus, the rise in PAP during the 20th min of exercise averaged 8.7 +/- 3.4 mm Hg after almitrine treatment while PAP increased by only 1.3 +/- 1.7 mm Hg before medication. The exaggerated exercise-induced PAP response in conjunction with the enhanced secretion of norepinephrine that we observed during almitrine treatment suggests that catecholamine could be involved in the pulmonary haemodynamic adjustments. Furthermore, mixed-venous PO2 (PvO2) both during rest and exercise declined with the prolongation of almitrine ingestion, suggesting that PvO2 might possibly be implicated in the pulmonary haemodynamic response to almitrine, in the same way as it is involved in the hypoxia-induced pulmonary vasoconstriction. These findings demonstrate that almitrine medication, even at a high dose, does not have any deleterious effect on pulmonary vasculature in resting conditions, but prolonged submaximal exercise should be proscribed in patients on a long-term therapy.