Vincent Joseph

Erythropoietin regulates hypoxic ventilation in mice by interacting with brainstem and carotid bodies

Apart from its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions in brain, retina and heart upon ischaemic injury. However, the physiological non‐erythroid functions of Epo remain unclear. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate Epos impact on ventilation upon hypoxic exposure. Tg21 mice showed improved ventilatory response to severe acute hypoxia and moreover improved ventilatory acclimatization to chronic hypoxic exposure. Furthermore, following bilateral transection of carotid sinus nerves that uncouples the brain from the carotid body, Tg21 mice adapted their ventilation to acute severe hypoxia while chemodenervated wild‐type (WT) animals developed a life‐threatening apnoea. These results imply that Epo in brain modulates ventilation. Additional analysis revealed that the Epo receptor (EpoR) is expressed in the main brainstem respiratory centres and suggested that Epo stimulates breathing control by alteration of catecholaminergic metabolism in brainstem. The modulation of hypoxic pattern of ventilation after i.v. injection of recombinant human Epo in WT mice and the dense EpoR immunosignal observed in carotid bodies showed that these chemoreceptors are sensitive to plasma levels of Epo. In summary, our results suggest that Epo controls ventilation at the central (brainstem) and peripheral (carotid body) levels. These novel findings are relevant to understanding better respiratory disorders including those occurring at high altitude.

Chronic intermittent hypoxia reduces ventilatory long-term facilitation and enhances apnea frequency in newborn rats

Ventilatory long-term facilitation (LTF; defined as gradual increase of minute ventilation following repeated hypoxic exposures) is well described in adult mammals and is hypothesized to be a protective mechanism against apnea. In newborns, LTF is absent during the first postnatal days, but its precise developmental pattern is unknown. Accordingly, this study describes this pattern of postnatal development. Additionally, we tested the hypothesis that chronic intermittent hypoxia (CIH) from birth alters this development. LTF was estimated in vivo using whole body plethysmography by exposing rat pups at postnatal days 1, 4, and 10 (P1, P4, and P10) to 10 brief hypoxic cycles (nadir 5% O2) and respiratory recordings during the following 2 h (recovery, 21% O2). Under these conditions, ventilatory LTF (gradual increase of minute ventilation during recovery) was clearly expressed in P10 rats but not in P1 and P4. In a second series of experiments, rat pups were exposed to CIH during the first 10 postnatal days (6 brief cyclic exposures at 5% O2 every 6 min followed by 1 h under normoxia, 24 h a day). Compared with P10 control rats, CIH enhanced hypoxic ventilatory response (estimated during the hypoxic cycles) specifically in male rat pups. Ventilatory LTF was drastically reduced in P10 rats exposed to CIH, which was associated with higher apnea frequency during recovery. We conclude that CIH from birth enhances hypoxic chemoreflex and disrupts LTF development, thus likely contributing to increase apnea frequency.

Soluble erythropoietin receptor is present in the mouse brain and is required for the ventilatory acclimatization to hypoxia.

While erythropoietin (Epo) and its receptor (EpoR) have been widely investigated in brain, the expression and function of the soluble Epo receptor (sEpoR) remain unknown. Here we demonstrate that sEpoR, a negative regulator of Epos binding to the EpoR, is present in the mouse brain and is down‐regulated by 62% after exposure to normobaric chronic hypoxia (10% O2 for 3 days). Furthermore, while normoxic minute ventilation increased by 58% in control mice following hypoxic acclimatization, sEpoR infusion in brain during the hypoxic challenge efficiently reduced brain Epo concentration and abolished the ventilatory acclimatization to hypoxia (VAH). These observations imply that hypoxic downregulation of sEpoR is required for adequate ventilatory acclimatization to hypoxia, thereby underlying the function of Epo as a key factor regulating oxygen delivery not only by its classical activity on red blood cell production, but also by regulating ventilation.

Chronic corticosterone elevation and sex‐specific augmentation of the hypoxic ventilatory response in awake rats

Perinatal stress disrupts normal development of the hypothalamo‐pituitary‐adrenal (HPA) axis. Adult male (but not female) rats previously subjected to a stress such as neonatal maternal separation (NMS) are characterized by chronic elevation of plasma corticosterone (Cort) levels and an abnormally elevated hypoxic ventilatory response through mechanisms that remain unknown. The present study tested the hypothesis that a chronic increase of plasma Cort levels alone augments the ventilatory response to hypoxia in adult rats. Three groups of Sprague–Dawley male and female rats were used (control, placebo and Cort implants). Rats subjected to chronic Cort elevation received a subcutaneous Cort implant (300 mg) 14 days prior to ventilatory measurements, whereas sham‐operated rats received placebo implants. Controls received no treatment. Plasma Cort levels and body weight profiles were measured to assess protocol efficiency. Whole body plethysmography was used to measure ventilatory activity and metabolic indices during normoxia and following a 20 min period of moderate hypoxia (12% O2). Male rats implanted with Cort showed a ventilatory response to hypoxia higher than placebo‐treated rats; this effect was mainly due to a larger tidal volume response. In females, Cort treatment increased the breathing frequency response but the effect on minute ventilation was not significant. Taken together, these data show that chronic elevation of Cort alone increases the ventilatory response to hypoxia, but in a sex‐specific manner. These data raise important questions regarding the mechanisms underlying the sexual dimorphism of this effect and the potential link between HPA axis dysfunction and respiratory disorders related to abnormal ventilatory chemoreflex.

Progesterone increases hypoxic ventilatory response and reduces apneas in newborn rats

We hypothesized that progesterone may enhance the hypoxic ventilatory response and reduce the occurrence of apneas in newborn male rats. We studied 10-day-old rats chronically exposed to progesterone (Prog) or vehicle through the milk of lactating mothers. Respiratory and metabolic recordings were performed using whole body plethysmography under normoxia and during hypoxic exposure (10% O(2)--30 min). While progesterone did not alter baseline breathing and metabolic rate, it increased hypoxic ventilatory response particularly by limiting the magnitude of the ventilatory roll-off during the second phase of the hypoxic ventilatory response (i.e. following 5 min of exposure). In parallel, progesterone lowered the number of spontaneous apneas and drastically reduced the occurrence of post-sigh apneas during hypoxic exposure by limiting the time of the post-sigh expiratory pause. Following domperidone injection (used to block peripheral D2 dopamine receptor), minute ventilation increased in Veh pups and the number of spontaneous apneas decreased. These responses were not observed in Prog pups, suggesting that progesterone reduces peripheral dopaminergic inhibition on breathing. We conclude that progesterone is a potent stimulant of hypoxic ventilatory response in newborn rats and effectively reduces the occurrence of apneas.

Caffeine Reduces Apnea Frequency and Enhances Ventilatory Long-Term Facilitation in Rat Pups Raised in Chronic Intermittent Hypoxia

The mechanisms underlying the therapeutic function of caffeine on apneas in preterm neonates are not well determined. To better understand these effects, we exposed rat pups from postnatal d 3–12 to chronic intermittent hypoxia (5% O2/100 s every 10 min; 6 cycles/h followed by 1 h at 21% O2, 24 h/d), a model mimicking hypoxemic exposure in apneic neonates. Then, using whole-body plethysmography, we evaluated minute ventilation, apnea frequency, and duration after i.p injection of caffeine citrate (20 mg/kg) or saline under normoxia and in response to either sustained (FiO2 12%, 20 min) or brief (FiO2 5%, 60 s, total 10 episodes of 8 min each) hypoxia. These tests were used to assess peripheral and central components of hypoxic response. The latter also assessed the ventilatory long-term facilitation during recovery (2 h). Caffeine injection increased minute ventilation under baseline and during recovery. This effect was correlated with a decrease in apnea frequency (not duration). On the contrary, caffeine did not change the ventilatory response to sustained or brief hypoxic exposure. These results suggest that the effects of caffeine on apnea depend on increased central normoxic respiratory drive and enhancement of ventilatory long-term facilitation rather than on higher hypoxic ventilatory response.

Breathing at high altitude

Acclimatization to long-term hypoxia takes place at high altitude and allows gradual improvement of the ability to tolerate the hypoxic environment. An important component of this process is the hypoxic ventilatory acclimatization (HVA) that develops over several days. HVA reveals profound cellular and neurochemical re-organization occurring both in the peripheral chemoreceptors and in the central nervous system (in brainstem respiratory groups). These changes lead to an enhanced activity of peripheral chemoreceptor and re-inforce the central translation of peripheral inputs to efficient respiratory motor activity under the steady low O2 pressure. We will review the cellular processes underlying these changes with a particular emphasis on changes of neurotransmitter function and ion channel properties in peripheral chemoreceptors, and present evidence that low O2 level acts directly on brainstem nuclei to induce cellular changes contributing to maintain a high tonic respiratory drive under chronic hypoxia. (This study is part of a multi-author review.)

Developmental profile of cholinergic and purinergic traits and receptors in peripheral chemoreflex pathway in cats

This study describes the developmental profile of specific aspects of cholinergic and purinergic neurotransmission in key organs of the peripheral chemoreflex: the carotid body (CB), petrosal ganglion (PG) and superior cervical ganglion (SCG). Using real time RT-PCR and Western blot analyses, we assessed both mRNA and protein expression levels for choline-acetyl-transferase (ChAT), nicotinic receptor (subunits alpha3, alpha4, alpha7, and beta2), ATP and purinergic receptors (P2X2 and P2X3). These analyses were performed on tissue from 1- and 15-day-old, 2-month-old, and adult cats. During development, ChAT protein expression level increased slightly in CB; however, this increase was more important in PG and SCG. In CB, mRNA level for alpha4 nicotinic receptor subunit decreased during development (90% higher in 1-day-old cats than in adults). In the PG, mRNA level for beta2 nicotinic receptor subunit increased during development (80% higher in adults than in 1-day-old cats). In SCG, mRNA for alpha7 nicotinic receptor levels increased (400% higher in adults vs. 1-day-old cats). Conversely, P2X2 receptor protein level was not altered during development in CB and decreased slightly in PG; a similar pattern was observed for the P2X3 receptor. Our findings suggest that in cats, age-related changes in cholinergic and purinergic systems (such as physiological expression of receptor function) are significant within the afferent chemoreceptor pathway and likely contribute to the temporal changes of O2-chemosensitivity during development.

Neurochemical perspectives on the control of breathing during sleep

A specific depression of minute ventilation occurs during sleep in normal subjects. This sleep-related ventilatory depression is partially related to mechanical events and upper airway atonia but some data also indicate that it is likely to be centrally mediated. This paper reviews the anatomical and neurochemical connections between sleep/wake- and respiratory-related areas in an attempt to identify the potential implication of sleep-related neurochemicals (serotonin, catecholamines, GABA, acetylcholine) in the sleep-related hypoventilation. The review of available data suggests that the sleep-related ventilatory depression depends upon the enhanced GABAergic activity together with a loss of suprapontine influence depending on the cessation of activity of the reticular formation. During REM sleep, an additional inhibitory activity emerges from the pontine cholinergic neurons, which contributes to the breathing irregularities and the associated depression of minute ventilation and ventilatory response to chemical stimuli. This model may contribute to a better understanding of the neurochemical environment of respiratory neurons during sleep, which remains a question of importance regarding the numerous pathological states that are linked to specific perturbations of breathing control during sleep.

Expression of sex-steroid receptors and steroidogenic enzymes in the carotid body of adult and newborn male rats.

This study describes the localization and pattern of expression of estradiol and progesterone receptors as well as key enzymes for steroid synthesis (i.e. P450 side-chain-cleavage--P450scc, and P450 aromatase--P450Aro) in the carotid body (CB) and superior cervical ganglion (SCG) of adult, newborn and late fetal male rats, using immunohistochemistry, Western blot and real-time RT-PCR. Our results show a constitutive expression of the beta estradiol receptor (Erbeta) and the 80 kDa and 60 kDa progesterone receptors (PR-A and PR-C) isoforms in the CB, while in the SCG Eralpha, Erbeta, PR-A and PR-C are expressed. While P450Aro staining was negative, P450scc staining was strong both in the SCG and CB. In late fetal and newborn rats, Eralpha was not detected in the CB or SCG, but a slight staining appeared for P450 aromatase in the CB, and to a lesser extent in SCG. P450scc was strongly expressed in CB and SCG of late fetal and newborn rats. We conclude that the carotid body shows a constitutive expression of Erbeta and PR and may be able to synthesize steroids, including estradiol during late fetal life.