In Transgenic Erythropoietin Deficient Mice, an Increase in Respiratory Response to Hypercapnia Parallels Abnormal Distribution of CO2/H+-Activated Cells in the Medulla Oblongata.

Erythropoietin (Epo) and its receptor are expressed in central respiratory areas. We hypothesized that chronic Epo deficiency alters functioning of central respiratory areas and thus the respiratory adaptation to hypercapnia. The hypercapnic ventilatory response (HcVR) was evaluated by whole body plethysmography in wild type (WT) and Epo deficient (Epo-TAgh) adult male mice under 4%CO2. Epo-TAgh mice showed a larger HcVR than WT mice because of an increase in both respiratory frequency and tidal volume, whereas WT mice only increased their tidal volume. A functional histological approach revealed changes in CO2/H+-activated cells between Epo-TAgh and WT mice. First, Epo-TAgh mice showed a smaller increase under hypercapnia in c-FOS-positive number of cells in the retrotrapezoid nucleus/parafacial respiratory group than WT, and this, independently of changes in the number of PHOX2B-expressing cells. Second, we did not observe in Epo-TAgh mice the hypercapnic increase in c-FOS-positive number of cells in the nucleus of the solitary tract present in WT mice. Finally, whereas hypercapnia did not induce an increase in the c-FOS-positive number of cells in medullary raphe nuclei in WT mice, chronic Epo deficiency leads to raphe pallidus and magnus nuclei activation by hyperacpnia, with a significant part of c-FOS positive cells displaying an immunoreactivity for serotonin in the raphe pallidus nucleus. All of these results suggest that chronic Epo-deficiency affects both the pattern of ventilatory response to hypercapnia and associated medullary respiratory network at adult stage with an increase in the sensitivity of 5-HT and non-5-HT neurons of the raphe medullary nuclei leading to stimulation of f R for moderate level of CO2.

Effect of exercise training in rats exposed to chronic hypoxia: Application for Monge's disease.

Physical exercise may improve hematological conditions in high altitude dwellers suffering from Chronic Mountain Sickness (CMS), in reducing hemoglobin concentration. Therefore, the present study aimed to characterize the effects of 1-month exercise training session in a model of rats exposed to chronic hypoxia. Four groups of male rats were studied: normoxic sedentary (NS, n = 8), normoxic training (NT, n = 8), hypoxic sedentary (HS, n = 8), and hypoxic training group (HT, n = 8). Hypoxic groups were exposed to hypobaric hypoxia for one month (PB =433 Torr). Training intensity was progressively increased from a running speed of 10.4 to 17.8 m/min. Chronic hypoxia led to an increase in hematocrit (HCT) associated with a decrease in plasma volume despite an increase in water intake. Training led to a reduction in HCT (p < 0.01), with a non-significant increase in plasma volume and weight gain. Hypoxia and training had inhibitory effects on haptoglobin (NS group: 379 ± 92; HT: 239 ± 34 µg/ml, p < 0.01). Chronic hypoxia and exercise training increased SpO2 measured after acute hypoxic exposure. Training blunted the decrease in V˙ O2 peak, time of exhaustion, and maximum speed associated with chronic exposure to hypoxia. Chronic hypoxia led to a right ventricular hypertrophy, which was not corrected by 1-month exercise training. Altogether, by decreasing hematocrit, reducing body weight, and limiting performance decrease, training in hypoxia may have a beneficial effect on excessive erythropoiesis in chronic hypoxia. Therefore, regular exercise training might be beneficial to avoid worsening of CMS symptoms in high altitude dwellers and to improve their quality of life.

Acute Effects of Systemic Erythropoietin Injections on Carotid Body Chemosensory Activity Following Hypoxic and Hypercapnic Stimulation.

The carotid body (CB) chemoreceptors sense changes in arterial blood gases. Upon stimulation CB chemoreceptors cells release one or more transmitters to excite sensory nerve fibers of the carotid sinus nerve. While several neurotransmitters have been described to contribute to the CB chemosensory process less is known about modulatory molecules. Recent data suggest that erythropoietin (Epo) is involved in the control of ventilation, and it has been shown that Epo receptor is constitutively expressed in the CB chemoreceptors, suggesting a possible role for Epo in regulation of CB function. Therefore, in the present study we aimed to determine whether exogenous applications of Epo modulate the hypoxic and hypercapnic CB chemosensory responses. Carotid sinus nerve discharge was recorded in-situ from anesthetized adult male and female Sprague Dawley rats (350 g, n = 8) before and after systemic administration of Epo (2000 UI/kg). CB-chemosensitivity to hypoxia and hypercapnia was calculated by exposing the rat to FiO2 5-15% and FiCO2 10% gas mixtures, respectively. During baseline recordings at normoxia, we found no effects of Epo on CB activity both in male and female rats. In addition, Epo had no effect on maximal CB response to hypoxia in both male and female rats. Epo injections enhanced the maximum CB chemosensory response to hypercapnia in female rats (before vs. after Epo, 72.5 ± 7.1 Hz vs. 108.3 ± 6.9 Hz, p < 0.05). In contrast, Epo had no effect on maximum CB chemosensory response to hypercapnia in male rats but significantly increased the response recovery times (time required to return to baseline discharge following hypercapnic stimulus) from 2.1 ± 0.1 s to 8.2 ± 2.3 s (p < 0.05). Taken together, our results suggest that Epo has some modulatory effect on the CB chemosensory response to hypercapnia.

Effect of Gender on Chronic Intermittent Hypoxic Fosb Expression in Cardiorespiratory-Related Brain Structures in Mice.

We aimed to delineate sex-based differences in neuroplasticity that may be associated with previously reported sex-based differences in physiological alterations caused by repetitive succession of hypoxemia-reoxygenation encountered during obstructive sleep apnea (OSA). We examined long-term changes in the activity of brainstem and diencephalic cardiorespiratory neuronal populations induced by chronic intermittent hypoxia (CIH) in male and female mice by analyzing Fosb expression. Whereas the overall baseline and CIH-induced Fosb expression in females was higher than in males, possibly reflecting different neuroplastic dynamics, in contrast, structures responded to CIH by Fosb upregulation in males only. There was a sex-based difference at the level of the rostral ventrolateral reticular nucleus of the medulla, with an increase in the number of FOSB/ΔFOSB-positive cells induced by CIH in males but not females. This structure contains neurons that generate the sympathetic tone and which are involved in CIH-induced sustained hypertension during waking hours. We suggest that the sex-based difference in neuroplasticity of this structure contributes to the reported sex-based difference in CIH-induced hypertension. Moreover, we highlighted a sex-based dimorphic phenomenon in serotoninergic systems induced by CIH, with increased serotoninergic immunoreactivity in the hypoglossal nucleus and a decreased number of serotoninergic cells in the dorsal raphe nucleus in male but not female mice. We suggest that this dimorphism in the neuroplasticity of serotoninergic systems predisposes males to a greater alteration of neuronal control of the upper respiratory tract associated with the greater collapsibility of upper airways described in male OSA subjects.

Increased ventilation in female erythropoietin-deficient mouse line is not progesterone and estrous stage-dependent.

Previous studies suggest that chronic erythropoietin (Epo) deficiency in male mice does not alter normoxic/hypoxic ventilation. As effects of Epo are sex specific and as progesterone could be a respiratory stimulant, we evaluated the impact of Epo deficiency and its possible interaction with progesterone in ventilatory control in female mice during estrous cycle phases. Compared to wild type (WT) animals, Epo-TAgh female mice exhibited higher ventilation in hypoxia. However, when data were separated into luteal and follicular phases of the estrous cycle, basal ventilation and hypoxic ventilation were not different in both mice strains. As progesterone is known to be a potent respiratory stimulant, additional experiments were performed to elucidate its role. Interestingly, after mifepristone treatment, HVR was not modified in WT and Epo-TAgh mice, showing that the ventilatory stimulation observed in females was not directly mediated by progesterone. We conclude that Epo-TAgh female mice show no estrous stage-dependent increase of ventilatory control and progesterone independent response to hypoxia.

Pharmacological, but not genetic, alteration of neural Epo modifies the CO2/H+ central chemosensitivity in postnatal mice.

Cerebral erythropoietin (Epo) plays a crucial role for respiratory control in newborn rodents. We showed previously that soluble Epo receptor (sEpoR: an Epo antagonist) reduces basal ventilation and hypoxic hyperventilation at postnatal day 10 (P10) and in adult mice. However, at these ages (P10 and adulthood), Epo had no effect on central chemosensitivity. Nevertheless, it is known that the sensitivity to CO2/H+ during the mammalian respiratory network maturation process is age-dependent. Accordingly, in this study we wanted to test the hypothesis that cerebral Epo is involved in the breathing stimulation induced by the activation of central CO2/H+ chemoreceptors at earlier postnatal ages. To this end, en bloc brainstem-spinal cord preparations were obtained from P4 mice and the fictive breathing response to CO2-induced acidosis or metabolic acidosis was analyzed. This age (P4) was chosen because previous research from our laboratory showed that Epo altered (in a dose- and time-dependent manner) the fictive ventilation elicited in brainstem-spinal cord preparations. Moreover, as it was observed that peripheral chemoreceptors determined the respiratory sensitivity of central chemoreceptors to CO2, the use of this technique restricts our observations to central modulation. Our results did not show differences between preparations from control and transgenic animals (Tg21: overexpressing cerebral Epo; Epo-TAgh: cerebral Epo deficient mice). However, when Tg21 brainstem preparations were incubated for 1h with sEpoR, or with inhibitors of ERK/Akt (thus blocking the activation of the Epo molecular pathway), the fictive breathing response to CO2-induced acidosis was blunted. Our data suggest that variation of the Epo/sEpoR ratio is central to breathing modulation during CO2 challenges, and calls attention to clinical perspectives based on the use of Epo drugs at birth in hypoventilation cases.

Erythropoietin and the use of a transgenic model of erythropoietin-deficient mice.

Despite its well-known role in red blood cell production, it is now accepted that erythropoietin (Epo) has other physiological functions. Epo and its receptors are expressed in many tissues, such as the brain and heart. The presence of Epo/Epo receptors in these organs suggests other roles than those usually assigned to this protein. Thus, the aim of this review is to describe the effects of Epo deficiency on adaptation to normoxic and hypoxic environments and to suggest a key role of Epo on main physiological adaptive functions. Our original model of Epo-deficient (Epo-TAgh) mice allowed us to improve our knowledge of the possible role of Epo in O2 homeostasis. The use of anemic transgenic mice revealed Epo as a crucial component of adaptation to hypoxia. Epo-TAgh mice survive well in hypoxic conditions despite low hematocrit. Furthermore, Epo plays a key role in neural control of ventilatory acclimatization and response to hypoxia, in deformability of red blood cells, in cerebral and cardiac angiogenesis, and in neuro- and cardioprotection.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

Comparative ventilatory strategies of acclimated rats and burrowing plateau pika (Ochotona curzoniae) in response to hypoxic-hypercapnia.

The objective of this study was to compare the different ventilatory strategies that help in coping with hypoxic-hypercapnia environment among two species: use acclimated rats and plateau pikas (Ochotona curzoniae) that live in Tibetan plateaus, and have been well adjusted to high altitude. Arterial blood samples taken at 4100 m of elevation in acclimatized rats and adapted pikas revealed inter-species differences with lower hemoglobin and hematocrit and higher blood pH in pikas. A linear and significant increase in minute ventilation was observed in pikas, which help them to cope with hypoxic-hypercapnia. Pikas also displayed a high inspiratory drive and an invariant respiratory timing regardless of the conditions. Biochemical analysis revealed that N-methyl-D-aspartate receptor (NMDA) receptor gene and nNOS gene are highly conserved between rats and pikas, however pikas have higher expression of NMDA receptors and nNOS compared to rats at the brainstem level. Taken together, these results suggest that pikas have developed a specific ventilatory pattern supported by a modification of the NMDA/NO ventilatory central pathways to survive in extreme conditions imposed on the Tibetan plateaus. These physiological adaptive strategies help in maintaining a better blood oxygenation despite high CO2 concentration in burrows at high altitude.

Role of glutamate and serotonin on the hypoxic ventilatory response in high-altitude-adapted plateau Pika.

The highland "plateau Pika" is considered to be adapted to chronic hypoxia. We hypothesized that glutamate N-methyl-D-aspartate (NMDA) and non-NMDA receptors, nitric oxide (NO) synthase, and serotonin are involved in hypoxic ventilatory response (HVR) in Pikas. We tested the effects of NMDA (memantine) and non-NMDA receptors (DNQX) antagonists, NO synthase inhibitor (L-NAME), and selective serotonin reuptake inhibitors (fluoxetine) on ventilation and HVR in Pikas. Ventilatory parameters were measured before and after drug (or vehicle) injections in conscious Pikas at their natural living altitude (PIO2 86 mmHg) and after a hypoxic challenge (PIO2 57 mmHg, 3 min) to assess the influence of peripheral chemoreceptor on HVR. Minute ventilation (VI) and tidal volume (Vt) increased during hypoxic challenge after vehicle injection, whereas the Ti/Ttot ratio remained unchanged. The increase in VI and Vt observed with vehicle at PIO2-57, when compared with PIO2-86, was inhibited after memantine and fluoxetine injection, whereas the DNQX injection increased HVR. At PIO2-57, L-NAME induced an increase in the Ti/Ttot ratio when compared with vehicle. Therefore, the glutamate through NMDA-R/AMPA receptor bindings and serotonin pathway are implicated at the peripheral chemoreceptor level in HVR in Pikas. However, NO influences the ventilatory pattern of Pikas at their habitual living altitude.

Catalyzing role of erythropoietin on the nitric oxide central pathway during the ventilatory responses to hypoxia.

The N-Methyl-d-Aspartate (NMDA) receptors - neuronal nitric oxide synthase (nNOS) pathway is involved in the ventilatory response to hypoxia. The objective was to assess the possible effect of erythropoietin deficiency and chronic exposure to hypoxia on this pathway during ventilatory response to acute hypoxia. Wild-type (WT) and erythropoietin-deficient (Epo-TAg(h)) male mice were exposed (14 days) either to hypobaric hypoxia (Pb = 435 mmHg) or to normoxia. The ventilation was measured at 21% or 8% O2 after injection of vehicle (NaCl), nNOS inhibitor (SMTC) or NMDA receptor antagonist (MK-801). Nitric oxide production and the expression of NMDA receptor and nNOS were assessed by real-time RT-PCR and Western blot analyses in the medulla. At rest, Epo-TAg(h) mice displayed normal ventilatory parameters at 21% O2 but did not respond to acute hypoxia despite a larger expression of NMDA receptors and nNOS in the medulla. Ventilatory acclimatization to hypoxia was observed in WT but was absent in Epo-TAg(h) mice. nNOS inhibition blunted the hypoxic ventilatory acclimatization of WT mice without any effect in Epo-TAg(h) mice. Acute hypoxic ventilatory response (HVR) was increased after chronic hypoxia in WT but remained unchanged in Epo-TAg(h) mice. Ventilatory response to acute hypoxia was modified by MK-801 injection in WT and Epo-TAg(h) mice. The results confirm that adequate erythropoietin level is necessary to obtain an appropriate HVR and a significant ventilatory acclimatization to hypoxia. Furthermore, erythropoietin plays a potential catalyzing role in the NMDA-NO central pathway during the ventilatory response and acclimatization to hypoxia.