Central CO2 chemosensitivity and CO2 controller gain independently contribute to daytime Pco2 in young subjects with congenital central hypoventilation syndrome.

Whether peripheral chemoreceptor response is altered in congenital central hypoventilation syndrome (CCHS) remains debated. Our aim was to prospectively evaluate both peripheral and central CO2 chemosensitivity and to evaluate their correlations with daytime Pco2 and arterial desaturation during exercise in CCHS. To this end, tidal breathing was recorded in patients with CCHS allowing the calculation of loop gain and its components {steady-state controller (assumed to mainly be peripheral chemosensitivity) and plant gains using a bivariate [end-tidal Pco2 ([Formula: see text]) and ventilation] constrained model}, a hyperoxic, hypercapnic ventilatory response test (central chemosensitivity), and a 6-min walk test (arterial desaturation). The results of loop gain were compared with those previously obtained in a healthy group of similar age. The study prospectively included 23 subjects with CCHS, without daytime ventilatory support; the subjects had a median age of 10 (5.6 to 27.4) yr (15 females) with moderate polyalanine repeat mutation (PARM: 20/25, 20/26, n = 11), severe PARM (20/27, 20/33, n = 8), or non-PARM (n = 4). As compared with 23 healthy subjects (4.9-27.0 yr), the subjects with CCHS had a decreased controller gain and an increased plant gain. Mean daytime [Formula: see text] level of subjects with CCHS correlated negatively to both Log(controller gain) and the slope of CO2 response. Genotype was not related to chemosensitivity. Arterial desaturation on exercise correlated negatively with Log(controller) gain but not with the slope of the CO2 response. In conclusion, we demonstrate that peripheral CO2 chemosensitivity is altered in some patients with CCHS and that the daytime [Formula: see text] depends on central and peripheral chemoreceptor responses.NEW & NOTEWORTHY Altered central CO2 chemosensitivity is a hallmark of congenital central hypoventilation syndrome (CCHS). Peripheral CO2 chemosensitivity can be partly assessed by controller gain measurement obtained from tidal breathing recording. In young subjects with CCHS, this study shows that both central and peripheral CO2 sensitivities independently contribute to daytime Pco2. Hypocapnia during nighttime-assisted ventilation is associated with higher peripheral chemosensitivity that is further associated with lesser arterial desaturation at walk.

A decrease in plant gain, namely CO2 stores, characterizes dysfunctional breathing whatever its subtype in children.

Background: Whether dysfunctional breathing (DB) subtype classification is useful remains undetermined. The hyperventilation provocation test (HVPT) is used to diagnose DB. This test begins with a 3-min phase of hyperventilation during which fractional end-tidal CO2 (FETCO2) decreases that could be an assessment of plant gain, which relies on CO2 stores. Our aim was to assess 1) whether the children suffering from different subtypes of DB exhibit decreased plant gain and 2) the relationships between HVPT characteristics and plant gain. Methods: We retrospectively selected 48 children (median age 13.5 years, 36 females, 12 males) who exhibited during a cardiopulmonary exercise test either alveolar hyperventilation (transcutaneous PCO2 < 30 mmHg, n = 6) or inappropriate hyperventilation (increased VE'/V'CO2 slope) without hypocapnia (n = 18) or dyspnea without hyperventilation (n = 18) compared to children exhibiting physiological breathlessness (dyspnea for sports only, n = 6). These children underwent tidal-breathing recording (ventilation and FETCO2 allowing the calculation of plant gain) and a HVPT. Results: The plant gain was significantly higher in the physiological group as compared to the dyspnea without hyperventilation group, p = 0.024 and hyperventilation without hypocapnia group, p = 0.008 (trend for the hyperventilation with hypocapnia group, p = 0.078). The slope of linear decrease in FETCO2 during hyperventilation was significantly more negative in physiological breathlessness group as compared to hyperventilation without hypocapnia group (p = 0.005) and dyspnea without hyperventilation group (p = 0.049). Conclusion: The children with DB, regardless of their subtype, deplete their CO2 stores (decreased plant gain), which may be due to intermittent alveolar hyperventilation, suggesting the futility of our subtype classification.

Neurogenic hypertension characterizes children with congenital central hypoventilation syndrome and is aggravated by alveolar hypoventilation during sleep.

OBJECTIVES: Autonomic nervous system (ANS) dysfunction characterizes congenital central hypoventilation syndrome (CCHS). The objectives were to describe ambulatory blood pressure monitoring (ABPM) of children with CCHS, to assess cardiac ANS dysfunction as compared with control participants and to search for relationships between ANS dysfunction and blood pressure (BP) or night-time PCO 2 measurements. METHODS: Retrospective study of ABPM of children with CCHS and case (CCHS)-control (healthy children) study of heart rate variability (HRV) indices obtained during polysomnography (wakefulness, nonrapid eye movement sleep, rapid eye movement sleep, and whole night). The HRV indices analyzed were low, high-frequency powers, low frequency/high frequency, and for the whole night, SD1/SD2. RESULTS: Twenty-four children with CCHS (14 girls) who underwent 81 ABPM (2-6/patient, 74 after 4 years) were included in the longitudinal study. Hypertension was evidenced in 29 of 45 (64%) ABPM made between 5 and 9 years of age as compared with 12 of 36 (33%) ABPM made between 10 and 17 years of age ( P  = 0.005). In the case-control study (12 pairs), as compared with control children, children with CCHS were characterized by a decreased HRV while awake, which was aggravated at night. In children with CCHS, at daytime, SBP percentiles positively correlated with low-frequency power ( R  = -0.82; P  = 0.001), while at night-time, SBP percentiles negatively correlated with SD1/SD2 ( R  = -0.79; P  = 0.010). The SD1/SD2 ratio also negatively correlated with median PCO 2 under mechanical ventilation ( R  = -0.69; P  = 0.013). CONCLUSION: Neurogenic hypertension is frequent in CCHS and correlates with ANS dysfunction, which also correlates with alveolar ventilation during mechanical ventilation.

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.

Heart rate variability in congenital central hypoventilation syndrome: relationships with hypertension and sinus pauses.

BACKGROUND: Autonomic nervous system (ANS) dysregulation has been described in congenital central hypoventilation syndrome (CCHS). The objectives were to describe heart rate variability (HRV) analyses in children suffering from CCHS both while awake and asleep and their relationships with both ambulatory blood pressure (BP) and ECG monitoring results. METHODS: This retrospective study enrolled children with CCHS (n = 33, median age 8.4 years, 18 girls) who had BP and ECG monitored during the same 24 h. From the latter, HRV analyses were obtained during daytime and nighttime. RESULTS: The prevalences of hypertension and sinus pauses were 33% (95% confidence interval [CI]: 18-52) and 18% (95% CI: 7-35), respectively. The decrease in systolic BP at night negatively correlated with an increase in very low frequency (VLF) and LF powers at night, and the longest RR interval positively correlated with daytime VLF and LF powers. Among the three groups of children (polyalanine repeat expansion mutation [PARM], moderate [20/25 and 20/26], severe [20/27 and 20/33], and non-PARMs), the prevalence of elevated BP or hypertension was different: in PARM subjects: 6/18 moderate, 7/9 severe versus 0/6 in non-PARM (p = 0.002). CONCLUSION: Modifications of cardiac ANS are associated with systemic hypertension and the occurrence of sinus pauses in CCHS. IMPACT: Children with congenital central hypoventilation syndrome (CCHS) exhibit an increased prevalence of hypertension and sinus pauses that are linked to cardiac autonomic nervous system dysfunction. Sinus pauses are the main manifestation of sinus nodal dysfunction in children with CCHS. The increased prevalence of hypertension, especially at nighttime, is a new finding in CCHS. Sinus nodal dysfunction can be due to the sole impairment of the cardiac autonomic nervous system. Ambulatory blood pressure and ECG monitoring are mandatory in patients with CCHS.

Oropharyngeal obstruction and respiratory system compliance are linked to ventilatory control parameters in pediatric obstructive sleep apnea syndrome.

Instable ventilatory control is an endotypic trait of obstructive sleep apnea syndrome (OSAS). This study aimed to evaluate the relationships between the anatomical compromise of the upper (oro- and naso-pharynx) and lower airways and ventilatory control (measured by chemical loop gain) in otherwise healthy children suffering from moderate to severe OSAS (apnea hypopnea index ≥ 5/hour). The children underwent ear, nose and throat examination, measurement of impedance of the respiratory system that allowed characterizing peripheral lung mechanics using the extended Resistance-Inertance-Compliance model. Physiologically constrained analytical model based on tidal breathing analysis allowed for the computation of steady-state plant gain, steady-state controller gain (CG0) and steady-state loop gain (LG0). Medium-frequency components of the feedback control system were then deduced. Fifty children (median age 11.2 years) were enrolled. Oropharyngeal obstruction was associated with decreased CG0 (0.6 [0.2; 1.0] vs 1.5 [0.5; 6.6] L.s- 1.mmHg- 1, p = 0.038) and LG0 (0.4 [0.2; 1.1] vs 1.2 [0.4; 9.3], p = 0.027), while nasal obstruction did not modify ventilatory control parameters. In a multivariate analysis Medium-Frequency PG was negatively related to minute ventilation and respiratory system compliance. Both upper (tonsil hypertrophy) and lower (compliance of respiratory system) airways are linked to ventilatory control in children with moderate to severe OSAS.

Obstructive Apneas in a Mouse Model of Congenital Central Hypoventilation Syndrome.

Rationale: Congenital central hypoventilation syndrome (CCHS) is characterized by life-threatening sleep hypoventilation and is caused by PHOX2B gene mutations, most frequently the PHOX2B27Ala/+ mutation, with patients requiring lifelong ventilatory support. It is unclear whether obstructive apneas are part of the syndrome. Objectives: To determine if Phox2b27Ala/+ mice, which present the main symptoms of CCHS and die within hours after birth, also express obstructive apneas, and to investigate potential underlying mechanisms. Methods: Apneas were classified as central, obstructive, or mixed by using a novel system combining pneumotachography and laser detection of abdominal movement immediately after birth. Several respiratory nuclei involved in airway patency were examined by immunohistochemistry and electrophysiology in brainstem-spinal cord preparations. Measurements and Main Results: The median (interquartile range) of obstructive apnea frequency was 2.3 (1.5-3.3)/min in Phox2b27Ala/+ pups versus 0.6 (0.4-1.0)/min in wild types (P < 0.0001). Obstructive apnea duration was 2.7 seconds (2.3-3.9) in Phox2b27Ala/+ pups versus 1.7 seconds (1.1-1.9) in wild types (P < 0.0001). Central and mixed apneas presented similar significant differences. In Phox2b27Ala/+ preparations, the hypoglossal nucleus had fewer (P < 0.05) and smaller (P < 0.01) neurons, compared with wild-type preparations. Importantly, coordination of phrenic and hypoglossal motor activities was disrupted, as evidenced by the longer and variable delay of hypoglossal activity with respect to phrenic activity onset (P < 0.001). Conclusions: The Phox2b27Ala/+ mutation predisposed pups not only to hypoventilation and central apneas, but also to obstructive and mixed apneas, likely because of hypoglossal dysgenesis. These results thus demand attention toward obstructive events in infants with CCHS.

Nebulized curcumin protects neonatal lungs from antenatal insult in rats.

Intrauterine growth restriction (IUGR) increases the risk of bronchopulmonary dysplasia (BPD), one of the major complications of prematurity. Antenatal low-protein diet (LPD) exposure in rats induces IUGR and mimics BPD-related alveolarization disorders. Peroxisome proliferator-activated receptor-γ (PPARγ) plays a key role in normal lung development and was found deregulated following LPD exposure. The objective of this article was to investigate the effects of nebulized curcumin, a natural PPARγ agonist, to prevent IUGR-related abnormal lung development. We studied rat pups antenatally exposed to an LPD or control diet (CTL) and treated with nebulized curcumin (50 mg/kg) or vehicle from postnatal (P) days 1 to 5. The primary readouts were lung morphometric analyses at P21. Immunohistochemistry (P21) and microarrays (P6 and P11) were compared within animals exposed to LPD versus controls, with and without curcumin treatment. Quantitative morphometric analyses revealed that LPD induced abnormal alveolarization as evidenced by a significant increase in mean linear intercept (MLI) observed in P21 LPD-exposed animals. Early curcumin treatment prevented this effect, and two-way ANOVA analysis demonstrated significant interaction between diet and curcumin both for MLI [F(1,39) = 12.67, P = 0.001] and radial alveolar count at P21 [F(1,40) = 6.065, P = 0.0182]. Immunohistochemistry for fatty acid binding protein 4 (FABP4), a major regulator of PPARγ pathway, showed a decreased FABP4+ alveolar cell density in LPD-exposed animals treated by curcumin. Transcriptomic analysis showed that early curcumin significantly prevented the activation of profibrotic pathways observed at P11 in LPD-exposed animals. Nebulized curcumin appears to be a promising strategy to prevent alveolarization disorders in IUGR rat pups, targeting pathways involved in lung development.

Cross-sectional case-control study of the relationships between pharyngeal compliance and heart rate variability indices in childhood obstructive sleep apnoea.

A combination of noradrenergic and antimuscarinic agents reduces the apnea-hypopnea index (AHI) in adult patients with obstructive sleep apnoea (OSA) via reduced upper airway collapsibility, suggesting that a shift in the sympathovagal balance improves OSA. The objectives of our present case-control study were to assess heart rate variability (HRV) indices in the stages of sleep in children with and without OSA to evaluate OSA-induced sleep HRV modifications and to assess whether increased collapsibility measured during wakefulness is associated with reduced sympathetic activity during non-rapid eye movement (NREM) sleep. Three groups of 15 children were matched by sex, age, z-score of body mass index and ethnicity: non-OSA (obstructive AHI [OAHI] <2 events/hr), mild (OAHI ≥2 to <5 events/hr) or moderate-severe (OAHI ≥5 events/hr) OSA. Pharyngeal compliance was measured during wakefulness using acoustic pharyngometry. HRV indices (time and frequency domain variables) were calculated on 5-min electrocardiography recordings from polysomnography during wakefulness, NREM and REM sleep in periods free of any event. As compared to children without OSA, those with OSA (n = 30) were characterised by increased compliance and no physiological parasympathetic tone increase in REM sleep. Children with increased pharyngeal compliance (n = 21) had a higher OAHI due to higher AHI in NREM sleep, whereas their sympathetic tone was lower than that of those with normal compliance (n = 24). In conclusion, children with increased pharyngeal compliance exhibit decreased sympathetic tone associated with increased AHI in NREM sleep. Therapeutics directed at sympathovagal balance modifications should be tested in childhood OSA.

Cross-sectional study of loop gain abnormalities in childhood obstructive sleep apnea syndrome.

STUDY OBJECTIVES: We aimed to assess ventilatory control in typically developing children with and without obstructive sleep apnea (OSA). METHODS: Otherwise healthy children referred for suspicion of OSA were recruited. In addition to polysomnography, we analyzed loop, controller and plant gains (ie, LG, CG, and PG), which reflect the stability of control, chemoreceptor sensitivity and the pulmonary control of blood gases in response to changes in ventilation, respectively, from tidal breathing recordings during wakefulness. Two bivariate (ventilation, end-tidal CO2: one unconstrained and one constrained) and one trivariate (plus end-tidal oxygen) unconstrained model were used to assess model consistency and oxygen chemosensitivity. RESULTS: In sum, 54 children (median age 11.6 years) were included. Children with OSA (n = 19, [obstructive apnea-hypopnea index] OAHI ≥2.h-1) had a higher plant gain compared with those without OSA (n = 35), and it was positively correlated with apnea hypopnea index (AHI) (r2 = 0.10, p < 0.020). The two models showed consistent results. The bivariate constrained model showed that children with OAHI ≥5.h-1 showed an increased steady-state plant gain compared with children with OAHI <5.h-1. The trivariate model did not show evidence of any abnormality of oxygen chemosensitivity. CONCLUSION: Plant gain may contribute to OSA pathophysiology in children, and therapies directed at its reduction should be tested.

Breathing under Anesthesia: A Key Role for the Retrotrapezoid Nucleus Revealed by Conditional Phox2b Mutant Mice.

BACKGROUND: Optimal management of anesthesia-induced respiratory depression requires identification of the neural pathways that are most effective in maintaining breathing during anesthesia. Lesion studies point to the brainstem retrotrapezoid nucleus. We therefore examined the respiratory effects of common anesthetic/analgesic agents in mice with selective genetic loss of retrotrapezoid nucleus neurons (Phox2b mice, hereafter designated "mutants"). METHODS: All mice received intraperitoneal ketamine doses ranging from 100 mg/kg at postnatal day (P) 8 to 250 mg/kg at P60 to P62. Anesthesia effects in P8 and P14 to P16 mice were then analyzed by administering propofol (100 and 150 mg/kg at P8 and P14 to P16, respectively) and fentanyl at an anesthetic dose (1 mg/kg at P8 and P14 to P16). RESULTS: Most mutant mice died of respiratory arrest within 13 min of ketamine injection at P8 (12 of 13, 92% vs. 0 of 8, 0% wild type; Fisher exact test, P < 0.001) and P14 to P16 (32 of 42, 76% vs. 0 of 59, 0% wild type; P < 0.001). Cardiac activity continued after terminal apnea, and mortality was prevented by mechanical ventilation, supporting respiratory arrest as the cause of death in the mutants. Ketamine-induced mortality in mutants compared to wild types was confirmed at P29 to P31 (24 of 36, 67% vs. 9 of 45, 20%; P < 0.001) and P60 to P62 (8 of 19, 42% vs. 0 of 12, 0%; P = 0.011). Anesthesia-induced mortality in mutants compared to wild types was also observed with propofol at P8 (7 of 7, 100% vs. 0 of 17,7/7, 100% vs. 0/17, 0%; P < 0.001) and P14 to P16 (8 of 10, 80% vs. 0 of 10, 0%; P < 0.001) and with fentanyl at P8 (15 of 16, 94% vs. 0 of 13, 0%; P < 0.001) and P14 to P16 (5 of 7, 71% vs. 0 of 11, 0%; P = 0.002). CONCLUSIONS: Ketamine, propofol, and fentanyl caused death by respiratory arrest in most mice with selective loss of retrotrapezoid nucleus neurons, in doses that were safe in their wild type littermates. The retrotrapezoid nucleus is critical to sustain breathing during deep anesthesia and may prove to be a pharmacologic target for this purpose.

Myelination induction by a histamine H3 receptor antagonist in a mouse model of preterm white matter injury.

Fifteen million babies are born preterm every year and a significant number suffer from permanent neurological injuries linked to white matter injury (WMI). A chief cause of preterm birth itself and predictor of the severity of WMI is exposure to maternal-fetal infection-inflammation such as chorioamnionitis. There are no neurotherapeutics for this WMI. To affect this healthcare need, the repurposing of drugs with efficacy in other white matter injury models is an attractive strategy. As such, we tested the efficacy of GSK247246, an H3R antagonist/inverse agonist, in a model of inflammation-mediated WMI of the preterm born infant recapitulating the main clinical hallmarks of human brain injury, which are oligodendrocyte maturation arrest, microglial reactivity, and hypomyelination. WMI is induced by mimicking the effects of maternal-fetal infection-inflammation and setting up neuroinflammation. We induce this process at the time in the mouse when brain development is equivalent to the human third trimester; postnatal day (P)1 through to P5 with i.p. interleukin-1ß (IL-1ß) injections. We initiated GSK247246 treatment (i.p at 7 mg/kg or 20 mg/kg) after neuroinflammation was well established (on P6) and it was administered twice daily through to P10. Outcomes were assessed at P10 and P30 with gene and protein analysis. A low dose of GSK247246 (7 mg/kg) lead to a recovery in protein expression of markers of myelin (density of Myelin Basic Protein, MBP & Proteolipid Proteins, PLP) and a reduction in macro- and microgliosis (density of ionising adaptor protein, IBA1 & glial fibrillary acid protein, GFAP). Our results confirm the neurotherapeutic efficacy of targeting the H3R for WMI seen in a cuprizone model of multiple sclerosis and a recently reported clinical trial in relapsing-remitting multiple sclerosis patients. Further work is needed to develop a slow release strategy for this agent and test its efficacy in large animal models of preterm infant WMI.

Comparison of methods of chemical loop gain measurement during tidal ventilation in awake healthy subjects.

The loop gain (LG) is defined as the ratio of a ventilatory response over the perturbation in ventilation, and it is used to analyze ventilatory control stability. The LG can be derived from minute ventilation (V̇e), end-tidal Pco2 ([Formula: see text]), and end-tidal Po2 ([Formula: see text]) values. Several methods of LG assessment have been developed, which have never been compared. We evaluated the computability, the short-term repeatability, and the agreement of six published (or slightly modified) models for LG determination. These models included three unconstrained autoregressive models, univariate (V̇e), bivariate (V̇e, [Formula: see text]), and trivariate (V̇e, [Formula: see text], and [Formula: see text]), and three analytical transfer function constrained models based on V̇e, V̇e and CO2-sensitivity, and V̇e and central and peripheral CO2 sensitivities, respectively. The models were tested with tidal breathing data in 37 awake healthy subjects (median age 35 yr; 23 women, 14 men). Modeling failed in 11, 0, and 0 subjects for the three unconstrained models, respectively, and 4, 1, and 9 subjects for the three constrained models, respectively. Bland and Altman analyses of the LG values in the medium frequency range of two separate recordings demonstrated good repeatability for four models, excluding univariate and trivariate unconstrained models. The four repeatable models gave LG values that were in agreement (medium frequency LG, median 0.100-0.210), although the constrained model based on V̇e systematically overestimated LG values. The variances explained by these models were ∼20%. In conclusion, model-based analyses of tidal breathing were performed with different approaches that gave comparable results for chemical LG and explained variance.NEW & NOTEWORTHY Several methods of chemical loop gain measurement have been published but never compared. We show that a better repeatability is obtained with analytical constrained models compared with autoregressive unconstrained models and that the repeatable models gave comparable results of loop gain, even if the calculation based on ventilation-only recording gave higher values than those obtained with both ventilation and end-tidal Pco2 recording. The explained variance of ventilation was similar whatever the model.

Acetylcholine Modulates the Hormones of the Growth Hormone/Insulinlike Growth Factor-1 Axis During Development in Mice.

Pituitary growth hormone (GH) and insulinlike growth factor (IGF)-1 are anabolic hormones whose physiological roles are particularly important during development. The activity of the GH/IGF-1 axis is controlled by complex neuroendocrine systems including two hypothalamic neuropeptides, GH-releasing hormone (GHRH) and somatostatin (SRIF), and a gastrointestinal hormone, ghrelin. The neurotransmitter acetylcholine (ACh) is involved in tuning GH secretion, and its GH-stimulatory action has mainly been shown in adults but is not clearly documented during development. ACh, together with these hormones and their receptors, is expressed before birth, and somatotroph cells are already responsive to GHRH, SRIF, and ghrelin. We thus hypothesized that ACh could contribute to the modulation of the main components of the somatotropic axis during development. In this study, we generated a choline acetyltransferase knockout mouse line and showed that heterozygous mice display a transient deficit in ACh from embryonic day 18.5 to postnatal day 10, and they recover normal ACh levels from the second postnatal week. This developmental ACh deficiency had no major impact on weight gain and cardiorespiratory status of newborn mice. Using this mouse model, we found that endogenous ACh levels determined the concentrations of circulating GH and IGF-1 at embryonic and postnatal stages. In particular, serum GH level was correlated with brain ACh content. ACh also modulated the levels of GHRH and SRIF in the hypothalamus and ghrelin in the stomach, and it affected the levels of these hormones in the circulation. This study identifies ACh as a potential regulator of the somatotropic axis during the developmental period.

Corrigendum: Protective effects of intermittent hypoxia on brain and memory in a mouse model of apnea of prematurity.

[This corrects the article on p. 313 in vol. 6, PMID: 26582992.].

Safety study of Ciprofloxacin in newborn mice.

Ciprofloxacin, a broad-spectrum antimicrobial agent belonging to the fluoroquinolone family, is prescribed off-label in infants less than one year of age. Ciprofloxacin is included in the European Medicines Agency priority list of off-patent medicinal products requiring evaluation in neonates. This evaluation is undergoing within the TINN (Treat Infections in Neonates) FP7 EU project. As part of the TINN project, the present preclinical study was designed to assess the potential adverse effects of Ciprofloxacin on neurodevelopment, liver and joints in mice. Newborn mice received subcutaneous Ciprofloxacin at 10, 30 and 100 mg/kg/day from 2 to 12 postnatal days. Peak plasma levels of Ciprofloxacin were in the range of levels measured in human neonates. We examined vital functions in vivo, including cardiorespiratory parameters and temperature, psychomotor development, exploratory behavior, arthro-, nephro- and hepato-toxic effects. We found no effect of Ciprofloxacin at 10 and 30 mg/kg/day. In contrast, administration at 100 mg/kg/day delayed weight gain, impaired cardiorespiratory and psychomotor development, caused inflammatory infiltrates in the connective tissues surrounding the knee joint, and moderately increased extramedullary hematopoiesis. The present study pleads for careful watching of cardiorespiratory and motor development in neonates treated with Ciprofloxacin, in addition to the standard surveillance of arthrotoxicity.

Protective effects of intermittent hypoxia on brain and memory in a mouse model of apnea of prematurity.

Apnea of prematurity (AOP) is considered a risk factor for neurodevelopmental disorders in children based on epidemiological studies. This idea is supported by studies in newborn rodents in which exposure to intermittent hypoxia (IH) as a model of AOP significantly impairs development. However, the severe IH used in these studies may not fully reflect the broad spectrum of AOP severity. Considering that hypoxia appears neuroprotective under various conditions, we hypothesized that moderate IH would protect the neonatal mouse brain against behavioral stressors and brain damage. On P6, each pup in each litter was randomly assigned to one of three groups: a group exposed to IH while separated from the mother (IH group), a control group exposed to normoxia while separated from the mother (AIR group), and a group of untreated unmanipulated pups left continuously with their mother until weaning (UNT group). Exposure to moderate IH (8% O2) consisted of 20 hypoxic events/hour, 6 h per day from postnatal day 6 (P6) to P10. The stress generated by maternal separation in newborn rodents is known to impair brain development, and we expected this effect to be smaller in the IH group compared to the AIR group. In a separate experiment, we combined maternal separation with excitotoxic brain lesions mimicking those seen in preterm infants. We analyzed memory, angiogenesis, neurogenesis and brain lesion size. In non-lesioned mice, IH stimulated hippocampal angiogenesis and neurogenesis and improved short-term memory indices. In brain-lesioned mice, IH decreased lesion size and prevented memory impairments. Contrary to common perception, IH mimicking moderate apnea may offer neuroprotection, at least in part, against brain lesions and cognitive dysfunctions related to prematurity. AOP may therefore have beneficial effects in some preterm infants. These results support the need for stratification based on AOP severity in clinical trials of treatments for AOP, to determine whether in patients with moderate AOP, these treatments are beneficial or deleterious.

Emotional disorders in adult mice heterozygous for the transcription factor Phox2b.

Phox2b is an essential transcription factor for the development of the autonomic nervous system. Mice carrying one invalidated Phox2b allele (Phox2b(+/-)) show mild autonomic disorders including sleep apneas, and impairments in chemosensitivity and thermoregulation that recover within 10days of postnatal age. Because Phox2b is not expressed above the pons nor in the cerebellum, this mutation is not expected to affect brain development and cognitive functioning directly. However, the transient physiological disorders in Phox2b(+/-) mice might impair neurodevelopment. To examine this possibility, we conducted a behavioral test battery of emotional, motor, and cognitive functioning in adult Phox2b(+/-) mice and their wildtype littermates (Phox2b(+/+)). Adult Phox2b(+/-) mice showed altered exploratory behavior in the open field and in the elevated plus maze, both indicative of anxiety. Phox2b(+/-) mice did not show cognitive or motor impairments. These results suggest that also mild autonomic control deficits may disturb long-term emotional development.