Umbilical cord-derived mesenchymal stromal cell therapy to prevent the development of neurodevelopmental disorders related to low birth weight.

Low birth weight (LBW) increases the risk of neurodevelopmental disorders (NDDs) such as attention-deficit/hyperactive disorder and autism spectrum disorder, as well as cerebral palsy, for which no prophylactic measure exists. Neuroinflammation in fetuses and neonates plays a major pathogenic role in NDDs. Meanwhile, umbilical cord-derived mesenchymal stromal cells (UC-MSCs) exhibit immunomodulatory properties. Therefore, we hypothesized that systemic administration of UC-MSCs in the early postnatal period may attenuate neuroinflammation and thereby prevent the emergence of NDDs. The LBW pups born to dams subjected to mild intrauterine hypoperfusion exhibited a significantly lesser decrease in the monosynaptic response with increased frequency of stimulation to the spinal cord preparation from postnatal day 4 (P4) to P6, suggesting hyperexcitability, which was improved by intravenous administration of human UC-MSCs (1 × 105 cells) on P1. Three-chamber sociability tests at adolescence revealed that only LBW males exhibited disturbed sociability, which tended to be ameliorated by UC-MSC treatment. Other parameters, including those determined via open-field tests, were not significantly improved by UC-MSC treatment. Serum or cerebrospinal fluid levels of pro-inflammatory cytokines were not elevated in the LBW pups, and UC-MSC treatment did not decrease these levels. In conclusion, although UC-MSC treatment prevents hyperexcitability in LBW pups, beneficial effects for NDDs are marginal.

Serotonin and the ventilatory effects of etonogestrel, a gonane progestin, in a murine model of congenital central hypoventilation syndrome.

Introduction: Congenital Central Hypoventilation Syndrome, a rare disease caused by PHOX2B mutation, is associated with absent or blunted CO2/H+ chemosensitivity due to the dysfunction of PHOX2B neurons of the retrotrapezoid nucleus. No pharmacological treatment is available. Clinical observations have reported non-systematic CO2/H+ chemosensitivity recovery under desogestrel. Methods: Here, we used a preclinical model of Congenital Central Hypoventilation Syndrome, the retrotrapezoid nucleus conditional Phox2b mutant mouse, to investigate whether etonogestrel, the active metabolite of desogestrel, led to a restoration of chemosensitivity by acting on serotonin neurons known to be sensitive to etonogestrel, or retrotrapezoid nucleus PHOX2B residual cells that persist despite the mutation. The influence of etonogestrel on respiratory variables under hypercapnia was investigated using whole-body plethysmographic recording. The effect of etonogestrel, alone or combined with serotonin drugs, on the respiratory rhythm of medullary-spinal cord preparations from Phox2b mutants and wildtype mice was analyzed under metabolic acidosis. c-FOS, serotonin and PHOX2B were immunodetected. Serotonin metabolic pathways were characterized in the medulla oblongata by ultra-high-performance liquid chromatography. Results: We observed etonogestrel restored chemosensitivity in Phox2b mutants in a non-systematic way. Histological differences between Phox2b mutants with restored chemosensitivity and Phox2b mutant without restored chemosensitivity indicated greater activation of serotonin neurons of the raphe obscurus nucleus but no effect on retrotrapezoid nucleus PHOX2B residual cells. Finally, the increase in serotonergic signaling by the fluoxetine application modulated the respiratory effect of etonogestrel differently between Phox2b mutant mice and their WT littermates or WT OF1 mice, a result which parallels with differences in the functional state of serotonergic metabolic pathways between these different mice. Discussion: Our work thus highlights that serotonin systems were critically important for the occurrence of an etonogestrel-restoration, an element to consider in potential therapeutic intervention in Congenital Central Hypoventilation Syndrome patients.

Short-term cognitive loading deteriorates breathing pattern and gas exchange in adult patients with congenital central hypoventilation syndrome.

Question: Human PHOX2B mutations result in life-threatening sleep-related hypoventilation (congenital central hypoventilation syndrome, CCHS). Most patients retain ventilatory activity when awake through a respiratory-related cortical network. We hypothesised that this need to mobilise cortical resources to breathe would lead to breathing-cognition interferences during cognitive loading. Patients and methods: Seven adult CCHS patients (five women; median age 21) performed standard neuropsychological tests (paced auditory serial addition test - calculation capacity, working memory, sustained and divided attention; trail making test - visuospatial exploration capacity, cognitive processing speed, attentional flexibility; Corsi block-tapping test - visuospatial memory, short-term memory, working memory) during unassisted breathing and under ventilatory support. Ventilatory variables and transcutaneous haemoglobin oxygen saturation were recorded. Cortical connectivity changes between unassisted breathing and ventilatory support were assessed using electroencephalographic recordings (EEG). Results: Baseline performances were lower than expected in individuals of this age. During unassisted breathing, cognitive loading coincided with increased breathing variability, and decreases in oxygen saturation inversely correlated with an increasing number of apnoeic cycles per minute (rho -0.46, 95% CI -0.76 to -0.06, p=0.01). During ventilatory support, cognitive tasks did not disrupt breathing pattern and were not associated with decreased oxygen saturation. Ventilatory support was associated with changes in EEG cortical connectivity but not with improved test performances. Conclusions: Acute cognitive loads induce oxygen desaturation in adult CCHS patients during unassisted breathing, but not under ventilatory support. This justifies considering the use of ventilatory support during mental tasks in CCHS patients to avoid repeated episodes of hypoxia.

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.

Diaphragmatic Activity and Respiratory Function Following C3 or C6 Unilateral Spinal Cord Contusion in Mice.

The majority of spinal cord injuries (SCIs) are cervical (cSCI), leading to a marked reduction in respiratory capacity. We aimed to investigate the effect of hemicontusion models of cSCI on both diaphragm activity and respiratory function to serve as preclinical models of cervical SCI. Since phrenic motoneuron pools are located at the C3-C5 spinal level, we investigated two models of preclinical cSCI mimicking human forms of injury, namely, one above (C3 hemicontusion-C3HC) and one below phrenic motoneuron pools (C6HC) in wild-type swiss OF-1 mice, and we compared their effects on respiratory function using whole-body plethysmography and on diaphragm activity using electromyography (EMG). At 7 days post-surgery, both C3HC and C6HC damaged spinal cord integrity above the lesion level, suggesting that C6HC potentially alters C5 motoneurons. Although both models led to decreased diaphragmatic EMG activity in the injured hemidiaphragm compared to the intact one (-46% and -26% in C3HC and C6HC, respectively, both p = 0.02), only C3HC led to a significant reduction in tidal volume and minute ventilation compared to sham surgery (-25% and -20% vs. baseline). Moreover, changes in EMG amplitude between respiratory bursts were observed post-C3HC, reflecting a change in phrenic motoneuronal excitability. Hence, C3HC and C6HC models induced alteration in respiratory function proportionally to injury level, and the C3HC model is a more appropriate model for interventional studies aiming to restore respiratory function in cSCI.

Serotonin 1A Receptor Pharmacotherapy and Neuroplasticity in Spinal Cord Injury.

Spinal cord injury is associated with damage in descending and ascending pathways between brainstem/cortex and spinal neurons, leading to loss in sensory-motor functions. This leads not only to locomotor reduction but also to important respiratory impairments, both reducing cardiorespiratory engagement, and increasing cardiovascular risk and mortality. Moreover, individuals with high-level injuries suffer from sleep-disordered breathing in a greater proportion than the general population. Although no current treatments exist to restore motor function in spinal cord injury (SCI), serotoninergic (5-HT) 1A receptor agonists appear as pharmacologic neuromodulators that could be important players in inducing functional improvements by increasing the activation of spared motoneurons. Indeed, single therapies of serotoninergic 1A (5-HT1A) agonists allow for acute and temporary recovery of locomotor function. Moreover, the 5-HT1A agonist could be even more promising when combined with other pharmacotherapies, exercise training, and/or spinal stimulation, rather than administered alone. In this review, we discuss previous and emerging evidence showing the value of the 5HT1A receptor agonist therapies for motor and respiratory limitations in SCI. Moreover, we provide mechanistic hypotheses and clinical impact for the potential benefit of 5-HT1A agonist pharmacology in inducing neuroplasticity and improving locomotor and respiratory functions in SCI.

Prenatal Hypoxia Induces Cl- Cotransporters KCC2 and NKCC1 Developmental Abnormality and Disturbs the Influence of GABAA and Glycine Receptors on Fictive Breathing in a Newborn Rat.

Prenatal hypoxia is a recognised risk factor for neurodevelopmental disorders associated with both membrane proteins involved in neuron homeostasis, e.g., chloride (Cl-) cotransporters, and alterations in brain neurotransmitter systems, e.g., catecholamines, dopamine, and GABA. Our study aimed to determine whether prenatal hypoxia alters central respiratory drive by disrupting the development of Cl- cotransporters KCC2 and NKCC1. Cl- homeostasis seems critical for the strength and efficiency of inhibition mediated by GABAA and glycine receptors within the respiratory network, and we searched for alterations of GABAergic and glycinergic respiratory influences after prenatal hypoxia. We measured fictive breathing from brainstem in ex vivo preparations during pharmacological blockade of KCC2 and NKCC1 Cl- cotransporters, GABAA, and glycine receptors. We also evaluated the membrane expression of Cl- cotransporters in the brainstem by Western blot and the expression of Cl- cotransporter regulators brain-derived neurotrophic factor (BDNF) and calpain. First, pharmacological experiments showed that prenatal hypoxia altered the regulation of fictive breathing by NKCC1 and KCC2 Cl- cotransporters, GABA/GABAA, and glycin. NKCC1 inhibition decreased fictive breathing at birth in control mice while it decreased at 4 days after birth in pups exposed to prenatal hypoxia. On the other hand, inhibition of KCC2 decreased fictive breathing 4 days after birth in control mice without any change in prenatal hypoxia pups. The GABAergic system appeared to be more effective in prenatal hypoxic pups whereas the glycinergic system increased its effectiveness later. Second, we observed a decrease in the expression of the Cl- cotransporter KCC2, and a decrease with age in NKCC1, as well as an increase in the expression of BDNF and calpain after prenatal hypoxia exposure. Altogether, our data support the idea that prenatal hypoxia alters the functioning of GABAA and glycinergic systems in the respiratory network by disrupting maturation of Cl- homeostasis, thereby contributing to long-term effects by disrupting ventilation.

Baclofen destabilises breathing during sleep in healthy humans: A randomised, controlled, double-blind crossover trial.

AIMS: Periodic breathing is frequent in patients with severe heart failure. Apart from being an indicator of severity, periodic breathing has its own deleterious consequences (sleep-related oxygen desaturations, sleep fragmentation), which justifies attempts to correct it irrespective of the underlying disease. Animal models and human data suggest that baclofen can reconfigure respiratory central pattern generators. We hypothesised that baclofen, a GABAB agonist, may thus be able to correct periodic breathing in humans. METHODS: Healthy volunteers were exposed to hypoxia during sleep. Participants who developed periodic breathing (n = 14 [53 screened]) were randomly assigned to double-blind oral baclofen (progressively increased to 60 mg/d) or placebo. The primary outcome was the coefficient of variation (CoVar) of respiratory cycle total time considered as an indicator of breathing irregularity. Secondary outcomes included the CoVar of tidal volume, apnoea-hypopnoea index, sleep fragmentation index and ventilatory complexity (noise limit). RESULTS: The analysis was conducted in 9 subjects after exclusion of incomplete datasets. CoVar of respiratory cycle total time significantly increased with baclofen during non-rapid eye movement sleep (median with placebo 56.00% [37.63-78.95]; baclofen 85.42% [68.37-86.40], P = .020; significant difference during the N1-N2 phases of sleep but not during the N3 phase). CoVar of tidal volume significantly increased during N1-N2 sleep. The apnoea-hypopnoea index, sleep fragmentation index and ventilatory complexity were not significantly different between placebo and baclofen. CONCLUSION: Baclofen did not stabilise breathing in our model. On the contrary, it increased respiratory variability. Baclofen should probably not be used in patients with or at risk of periodic breathing.

Topical treatment with a mu opioid receptor agonist alleviates corneal allodynia and corneal nerve sensitization in mice.

Corneal pain is considered to be a core symptom of ocular surface disruption and inflammation. The management of this debilitating condition is still a therapeutic challenge. Recent evidence supports a role of the opioid system in the management of corneal nociception. However, the functional involvement of the mu opioid receptor (MOR) underlying this analgesic effect is not known. We first investigated the expression of the MOR in corneal nerve fibers and trigeminal ganglion (TG) neurons in control mice and a mouse model of corneal inflammatory pain. We then evaluated the anti-nociceptive and electrophysiological effects of DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol] enkephalin), a MOR-selective ligand. MOR immunoreactivity was detected in corneal nerve fibers and primary afferent neurons of the ophthalmic branch of the TG of naive mice. MOR expression was significantly higher in both structures under conditions of inflammatory corneal pain. Topical ocular administration of DAMGO strongly reduced both the mechanical (von Frey) and chemical (capsaicin) corneal hypersensitivity associated with inflammatory ocular pain. Repeated instillations of DAMGO also markedly reversed the elevated spontaneous activity of the ciliary nerve and responsiveness of corneal polymodal nociceptors that were observed in mice with corneal pain. Finally, these DAMGO-induced behavioral and electrophysiological responses were totally blunted by the topical application of naloxone methiodide, an opioid receptor antagonist. Overall, these results provide evidence that topical pharmacological MOR activation may constitute a therapeutic target for the treatment of corneal pain and improve corneal nerve function to alleviate chronic pain.

Orexin Neurons Contribute to Central Modulation of Respiratory Drive by Progestins on ex vivo Newborn Rodent Preparations.

Dysfunction of central respiratory CO2/H+ chemosensitivity is a pivotal factor that elicits deep hypoventilation in patients suffering from central hypoventilation syndromes. No pharmacological treatment is currently available. The progestin desogestrel has been suggested to allow recovery of respiratory response to CO2/H+ in patients suffering from central hypoventilation, but except the fact that supramedullary regions may be involved, mechanisms are still unknown. Here, we tested in neonates whether orexin systems contribute to desogestrel's central effects on respiratory function. Using isolated ex vivo central nervous system preparations from newborn rats, we show orexin and almorexant, an antagonist of orexin receptors, supressed strengthening of the increase in respiratory frequency induced by prolonged metabolic acidosis under exposure to etonogestrel, the active metabolite of desogestrel. In parallel, almorexant suppressed the increase and enhanced increase in c-fos expression in respiratory-related brainstem structures induced by etonogestrel. These results suggest orexin signalisation is a key component of acidosis reinforcement of respiratory drive by etonogestrel in neonates. Although stage of development used is different as that for progestin clinical observations, presents results provide clues about conditions under which desogestrel or etonogestrel may enhance ventilation in patients suffering from central hypoventilation syndromes.

Neuromedin U is a gut peptide that alters oral glucose tolerance by delaying gastric emptying via direct contraction of the pylorus and vagal-dependent mechanisms.

The gut-brain peptide neuromedin U (NMU) decreases food intake and body weight and improves glucose tolerance. Here, we characterized NMU as an enteropeptide and determined how it impacts glucose excursion. NMU was expressed predominantly in the proximal small intestine, and its secretion was triggered by ingestion of a mixed meal. Although a single peripheral injection of NMU in C57BL/6NRj mice prevented the rise of glycemia upon an oral but not an intraperitoneal load of glucose, it unexpectedly prevented insulin secretion, only slightly improved peripheral insulin sensitivity, and barely reduced intestinal glucose absorption. Interestingly, peripheral administration of NMU abrogated gastric emptying. NMU receptors 1 and 2 were detected in pyloric muscles and NMU was able to directly induce pyloric contraction in a dose-dependent manner ex vivo in isometric chambers. Using a modified glucose tolerance test, we demonstrate that improvement of oral glucose tolerance by NMU was essentially, if not exclusively, because of its impact on gastric emptying. Part of this effect was abolished in vagotomized (VagoX) mice, suggesting implication of the vagus tone. Accordingly, peripheral injection of NMU was associated with increased number of c-FOS-positive neurons in the nucleus of the solitary tract, which was partly prevented in VagoX mice. Finally, NMU kept its ability to improve oral glucose tolerance in obese and diabetic murine models. Together, these data demonstrate that NMU is an enteropeptide that prevents gastric emptying directly by triggering pylorus contraction and indirectly through vagal afferent neurons. This blockade consequently reduces intestinal nutrient absorption and thereby results in an apparent improved tolerance to oral glucose challenge.-Jarry, A.-C., Merah, N., Cisse, F., Cayetanot, F., Fiamma, M.-N., Willemetz, A., Gueddouri, D., Barka, B., Valet, P., Guilmeau, S., Bado, A., Le Beyec, J., Bodineau, L., Le Gall, M. Neuromedin U is a gut peptide that alters oral glucose tolerance by delaying gastric emptying via direct contraction of the pylorus and vagal-dependent mechanisms.

Effects of corneal injury on ciliary nerve fibre activity and corneal nociception in mice: A behavioural and electrophysiological study.

BACKGROUND: Ocular surface diseases are among the most frequent ocular pathologies. Ocular pain following corneal injury is frequently observed in clinic. Corneal sensory innervation is supplied by ciliary nerves derived from ophthalmic division of the trigeminal ganglion. METHODS & RESULTS: Extracellular activity of the mouse ciliary nerve was first used to investigate the corneal responsiveness to chemical, mechanical and thermal stimulations in order to specifically study the responses of polymodal nociceptors, mechano-nociceptors and cold thermoreceptor in a control cornea. Then, in two models of corneal injury (repeated instillations of 0.02% benzalkonium chloride and corneal scraping), we first measured the corneal sensitivity to chemical (eye-wiping test) and mechanical (von Frey filaments) stimulation. Thereafter, we evaluated whether these corneal injuries modified the spontaneous and chemical stimulation-evoked activity of the ciliary nerve. Both models of injury induced a significant corneal chemical hypersensitivity correlated with an increase of the spontaneous activity of the ciliary nerve and a faster response of the ciliary nerve after a chemical stimulation. CONCLUSIONS: Overall, this study provides new insights into the functional aspects of corneal nerve fibre activity in mice after corneal injury. The increase in ciliary nerve activity may thus contribute to the development of ocular pain after corneal damage. SIGNIFICANCE: This study highlights the parallel increase in ciliary nerve activity and corneal sensitivity after corneal injury in mice. The strategy of combining ex vivo electrophysiological recordings of the ciliary nerve in mice and corneal sensitivity measurements therefore helps to uncover the functional aspects of corneal pain.

Mild Intrauterine Hypoperfusion Leads to Lumbar and Cortical Hyperexcitability, Spasticity, and Muscle Dysfunctions in Rats: Implications for Prematurity.

Intrauterine ischemia-hypoxia is detrimental to the developing brain and leads to white matter injury (WMI), encephalopathy of prematurity (EP), and often to cerebral palsy (CP), but the related pathophysiological mechanisms remain unclear. In prior studies, we used mild intrauterine hypoperfusion (MIUH) in rats to successfully reproduce the diversity of clinical signs of EP, and some CP symptoms. Briefly, MIUH led to inflammatory processes, diffuse gray and WMI, minor locomotor deficits, musculoskeletal pathologies, neuroanatomical and functional disorganization of the primary somatosensory and motor cortices, delayed sensorimotor reflexes, spontaneous hyperactivity, deficits in sensory information processing, memory and learning impairments. In the present study, we investigated the early and long-lasting mechanisms of pathophysiology that may be responsible for the various symptoms induced by MIUH. We found early hyperreflexia, spasticity and reduced expression of KCC2 (a chloride cotransporter that regulates chloride homeostasis and cell excitability). Adult MIUH rats exhibited changes in muscle contractile properties and phenotype, enduring hyperreflexia and spasticity, as well as hyperexcitability in the sensorimotor cortex. Taken together, these results show that reduced expression of KCC2, lumbar hyperreflexia, spasticity, altered properties of the soleus muscle, as well as cortical hyperexcitability may likely interplay into a self-perpetuating cycle, leading to the emergence, and persistence of neurodevelopmental disorders (NDD) in EP and CP, such as sensorimotor impairments, and probably hyperactivity, attention, and learning disorders.

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.

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.