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.

Current Perspectives for the use of Gonane Progesteronergic Drugs in the Treatment of Central Hypoventilation Syndromes.

BACKGROUND: Central alveolar hypoventilation syndromes (CHS) encompass neurorespiratory diseases resulting from congenital or acquired neurological disorders. Hypercapnia, acidosis, and hypoxemia resulting from CHS negatively affect physiological functions and can be lifethreatening. To date, the absence of pharmacological treatment implies that the patients must receive assisted ventilation throughout their lives. OBJECTIVE: To highlight the relevance of determining conditions in which using gonane synthetic progestins could be of potential clinical interest for the treatment of CHS. METHODS: The mechanisms by which gonanes modulate the respiratory drive were put into the context of those established for natural progesterone and other synthetic progestins. RESULTS: The clinical benefits of synthetic progestins to treat respiratory diseases are mixed with either positive outcomes or no improvement. A benefit for CHS patients has only recently been proposed. We incidentally observed restoration of CO2 chemosensitivity, the functional deficit of this disease, in two adult CHS women by desogestrel, a gonane progestin, used for contraception. This effect was not observed by another group, studying a single patient. These contradictory findings are probably due to the complex nature of the action of desogestrel on breathing and led us to carry out mechanistic studies in rodents. Our results show that desogestrel influences the respiratory command by modulating the GABAA and NMDA signaling in the respiratory network, medullary serotoninergic systems, and supramedullary areas. CONCLUSION: Gonanes show promise for improving ventilation of CHS patients, although the conditions of their use need to be better understood.

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.

Key Brainstem Structures Activated during Hypoxic Exposure in One-day-old Mice Highlight Characteristics for Modeling Breathing Network in Premature Infants.

We mapped and characterized changes in the activity of brainstem cell groups under hypoxia in one-day-old newborn mice, an animal model in which the central nervous system at birth is particularly immature. The classical biphasic respiratory response characterized by transient hyperventilation, followed by severe ventilation decline, was associated with increased c-FOS immunoreactivity in brainstem cell groups: the nucleus of the solitary tract, ventral reticular nucleus of the medulla, retrotrapezoid/parafacial region, parapyramidal group, raphe magnus nucleus, lateral, and medial parabrachial nucleus, and dorsal subcoeruleus nucleus. In contrast, the hypoglossal nucleus displayed decreased c-FOS immunoreactivity. There were fewer or no activated catecholaminergic cells activated in the medulla oblongata, whereas ~45% of the c-FOS-positive cells in the dorsal subcoeruleus were co-labeled. Approximately 30% of the c-FOS-positive cells in the parapyramidal group were serotoninergic, whereas only a small portion were labeled for serotonin in the raphe magnus nucleus. None of the c-FOS-positive cells in the retrotrapezoid/parafacial region were co-labeled for PHOX2B. Thus, the hypoxia-activated brainstem neuronal network of one-day-old mice is characterized by (i) the activation of catecholaminergic cells of the dorsal subcoeruleus nucleus, a structure implicated in the strong depressive pontine influence previously reported in the fetus but not in newborns, (ii) the weak activation of catecholaminergic cells of the ventral reticular nucleus of the medulla, an area involved in hypoxic hyperventilation, and (iii) the absence of PHOX2B-positive cells activated in the retrotrapezoid/parafacial region. Based on these results, one-day-old mice could highlight characteristics for modeling the breathing network of premature infants.

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.

Desogestrel enhances ventilation in ondine patients: Animal data involving serotoninergic systems.

Congenital central hypoventilation syndrome (CCHS) is a neurorespiratory disease characterized by life-threatening sleep-related hypoventilation involving an alteration of CO2/H(+) chemosensitivity. Incidental findings have suggested that desogestrel may allow recovery of the ventilatory response to CO2. The effects of desogestrel on resting ventilation have not been reported. This study was designed to test the hypothesis that desogestrel strengthens baseline ventilation by analyzing the ventilation of CCHS patients. Rodent models were used in order to determine the mechanisms involved. Ventilation in CCHS patients was measured with a pneumotachometer. In mice, ventilatory neural activity was recorded from ex vivo medullary-spinal cord preparations, ventilation was measured by plethysmography and c-fos expression was studied in medullary respiratory nuclei. Desogestrel increased baseline respiratory frequency of CCHS patients leading to a decrease in their PETCO2. In medullary spinal-cord preparations or in vivo mice, the metabolite of desogestrel, etonogestrel, induced an increase in respiratory frequency that necessitated the functioning of serotoninergic systems, and modulated GABAA and NMDA ventilatory regulations. c-FOS analysis showed the involvement of medullary respiratory groups of cell including serotoninergic neurons of the raphe pallidus and raphe obscurus nuclei that seem to play a key role. Thus, desogestrel may improve resting ventilation in CCHS patients by a stimulant effect on baseline respiratory frequency. Our data open up clinical perspectives based on the combination of this progestin with serotoninergic drugs to enhance ventilation in CCHS patients.