Effects of temperature on ventilatory response to hypercapnia in newborn mice heterozygous for transcription factor Phox2b.

Congenital central hypoventilation syndrome (CCHS) is a rare disease with variable severity, generally present from birth and chiefly characterized by impaired chemosensitivity to hypercapnia. The main cause of CCHS is a mutation in the PHOX2B gene, which encodes a transcription factor involved in the development of autonomic medullary reflex pathways. Temperature regulation is abnormal in many patients with CCHS. Here, we examined whether ambient temperature influenced CO(2) sensitivity in a mouse model of CCHS. A weak response to CO(2) at thermoneutrality (32 degrees C) was noted previously in 2-day-old mice with an invalidated Phox2b allele (Phox2b+/-), compared with wild-type littermates. We exposed Phox2b+/- pups to 8% CO(2) at three ambient temperatures (TAs): 29 degrees C, 32 degrees C, and 35 degrees C. We measured breathing variables and heart rate (HR) noninvasively using a novel whole body flow plethysmograph equipped with contact electrodes. Body temperature and baseline breathing increased similarly with TA in mutant and wild-type pups. The hypercapnic ventilatory response increased linearly with TA in both groups, while remaining smaller in mutant than in wild-type pups at all TAs. The differences between the absolute increases in ventilation in mutant and wild-type pups become more pronounced as temperature increased above 29 degrees C. The ventilatory abnormalities in mutant pups were not associated with significant impairments of heart rate control. In both mutant and wild-type pups, baseline HR increased with TA. In conclusion, TA strongly influenced the hypercapnic ventilatory response in Phox2b+/- mutant mice. These findings suggest that abnormal temperature regulation may contribute to the severity of respiratory impairments in CCHS patients.

Inhibitory effects of repeated hyperoxia on breathing in newborn mice.

Brief oxygen therapy is commonly used for resuscitation at birth or prevention of hypoxaemia before procedures during the neonatal period. However, O(2) may severely depress breathing, especially when administered repeatedly. The aim of the present study was to test the effects of repeated hyperoxia on breathing control in newborn mice. A total of 97 Swiss mouse pups were assigned to O(2) or air on post-natal day 0, 1 or 2. Each pup in the O(2) group was subjected to four hyperoxic tests (100% O(2) for 3 min followed by 12 min normoxia), whereas pups in the air group were maintained in normoxia. Breathing variables were measured using flow-through barometric plethysmography. O(2) significantly decreased minute ventilation as seen in a decrease in respiratory rate. This decrease became significantly larger with repeated exposure and ranged -17- -26% for all ages combined. Furthermore, hyperoxia increased total apnoea duration, as compared with the baseline value. In newborn mice, repeated hyperoxia increasingly depressed breathing. This finding further supports a need for stringent control of oxygen therapy, most notably repeated oxygen administration in the neonatal period for premature newborn infants and those carried to term.

Ventilatory response to hyperoxia in newborn mice heterozygous for the transcription factor Phox2b.

Heterozygous mutations of the transcription factor PHOX2B have been found in most patients with central congenital hypoventilation syndrome, a rare disease characterized by sleep-related hypoventilation and impaired chemosensitivity to sustained hypercapnia and sustained hypoxia. PHOX2B is a master regulator of autonomic reflex pathways, including peripheral chemosensitive pathways. In the present study, we used hyperoxic tests to assess the strength of the peripheral chemoreceptor tonic drive in Phox2b+/-newborn mice. We exposed 69 wild-type and 67 mutant mice to two hyperoxic tests (12-min air followed by 3-min 100% O2) 2 days after birth. Breathing variables were measured noninvasively using whole body flow plethysmography. The initial minute ventilation decrease was larger in mutant pups than in wild-type pups: -37% (SD 13) and -25% (SD 18), respectively, P<0.0001. Furthermore, minute ventilation remained depressed throughout O2 exposure in mutants, possibly because of their previously reported impaired CO2 chemosensitivity, whereas it returned rapidly to the normoxic level in wild-type pups. Hyperoxia considerably increased total apnea duration in mutant compared with wild-type pups (P=0.0001). A complementary experiment established that body temperature was not influenced by hyperoxia in either genotype group and, therefore, did not account for genotype-related differences in the hyperoxic ventilatory response. Thus partial loss of Phox2b function by heterozygosity did not diminish the tonic drive from peripheral chemoreceptors.

Automatic classification of activity and apneas using whole body plethysmography in newborn mice.

An increasing number of studies in newborn mice are being performed to determine the mechanisms of sleep apnea, which is the hallmark of early breathing disorders. Whole body plethysmography is the method of choice, as it does not require immobilization, which affects behavioral states and breathing. However, activity inside the plethysmograph may disturb the respiratory signal. Visual classification of the respiratory signal into ventilatory activity, activity-related disturbances, or apneas is so time-consuming as to considerably hamper the phenotyping of large pup samples. We propose an automatic classification of activity based on respiratory disturbances and of apneas based on spectral analysis. This method was validated in newborn mice on the day of birth and on postnatal days 2, 5, and 10, under normoxic and hypoxic (5% O(2)) conditions. For both activity and apneas, visual and automatic scores showed high Pearson's correlation coefficients (0.92 and 0.98, respectively) and high intraclass correlation coefficients (0.96-0.99), supporting strong agreement between the two methods. The present results suggest that breathing disturbances may provide a valid indirect index of activity in freely moving newborn mice and that automatic apnea classification based on spectral analysis may be efficient in terms of precision and of time saved.

Intermittent hypoxia induces transient arousal delay in newborn mice.

Previous studies suggested that defective arousal might be a major mechanism in sleep-disordered breathing such as sudden infant death syndrome and obstructive sleep apnea. In this study, we examined the effects of intermittent hypoxia (IH) on the arousal response to hypoxia in 4-day-old mice. We hypothesized that IH would increase arousal latency, as previously reported in other species, and we measured the concomitant changes in ventilation to shed light on the relationship between breathing and arousal. Arousal was scored according to behavioral criteria. Breathing variables were measured noninvasively by use of whole-body flow plethysmography. In the hypoxic group (n = 14), the pups were exposed to 5% O(2) in N(2) for 3 min and returned to air for 6 min. This test was repeated eight times. The normoxic mice (n = 14) were constantly exposed to normoxia. The hypoxic mice showed a 60% increase in arousal latency (P < 0.0001). Normoxic controls showed virtually no arousals. IH depressed normoxic ventilation below baseline prehypoxic levels, while preserving the ventilatory response to hypoxia. The breathing pattern and arousal responses recovered fully after 2 h of normoxia. We conclude that IH rapidly and reversibly depressed breathing and delayed arousal in newborn mice. Both effects may be due to hypoxia-induced release of inhibitory neurotransmitters acting concomitantly on both functions.

Classical conditioning of breathing pattern after two acquisition trials in 2-day-old mice.

The aim of the present study was to test whether breathing pattern conditioning may occur just after birth. We hypothesized that sensory stimuli signaling the resumption of maternal care after separation may trigger an arousal and/or orienting response accompanied with concomitant respiratory changes. We performed a conditioning experiment in 2-day-old mice by using an odor (lemon) as the conditioned stimulus (CS) and maternal care after 1 h without the mother as the unconditioned stimulus (US). Each pup underwent two acquisition trials, in which the CS was presented immediately before (experimental paired group, n = 30) or 30 min before (control unpaired group, n = 30) contact with the mother. Conditioning was tested by using noninvasive whole body plethysmography to measure the respiratory response to the CS for 1 min. We found significantly stronger respiratory responses to the CS in the experimental group than in the control group. In contrast, somatomotor activity did not differ significantly between groups. Our results confirm the sensitivity of breathing to conditioning and indirectly support the hypothesis that learned feedforward processes may complement feedback pathways during postnatal maturation of respiratory control.

Maturation of baseline breathing and of hypercapnic and hypoxic ventilatory responses in newborn mice.

Breathing during the first postnatal hours has not been examined in mice, the preferred mammalian species for genetic studies. We used whole body plethysmography to measure ventilation (VE), breath duration (T(TOT)), and tidal volume (VT) in mice delivered vaginally (VD) or by cesarean section (CS). In experiment 1, 101 VD and 100 CS pups aged 1, 6, 12, 24, or 48 h were exposed to 8% CO2 or 10% O2 for 90 s. In experiment 2, 31 VD pups aged 1, 12, or 24 h were exposed to 10% O2 for 5 min. Baseline breathing maturation was delayed in CS pups, but VE responses to hypercapnia and hypoxia were not significantly different between VD and CS pups [at postnatal age of 1 h (H1): 48 +/- 44 and 18 +/- 32%, respectively, in VD and CS pups combined]. The VE increase induced by hypoxia was greater at H12 (46 +/- 27%) because of T(TOT) response maturation. At all ages, hypoxic decline was ascribable mainly to a VT decrease, and posthypoxic decline was ascribable to a T(TOT) increase with apneas, suggesting different underlying neuronal mechanisms.

Impaired ventilatory responses to hypoxia in mice deficient in endothelin-converting-enzyme-1.

Endothelin-converting-enzyme (ECE-1) catalyzes the proteolytic activation of big endothelin-1 to mature endothelin-1. Most homozygous ECE-1-/- embryos die in utero and show severe craniofacial, enteric, and cardiac malformations precluding ventilatory function assessment. In contrast, heterozygous ECE-1+/- embryos develop normally. Their respiratory function at birth has not been studied. Taking into account previous respiratory investigations in mice with endothelin-1 gene disruption, we hypothesized that ECE-1-deficient mice may have impaired ventilatory control. We analyzed ventilatory responses to hypercapnia (8% CO(2)) and hypoxia (10% O(2)) in newborn and adult mice heterozygous for ECE-1 deficiency (ECE-1+/-) and in their wild-type littermates (ECE-1+/+). Ventilation, breath duration, and tidal volume were measured using whole-body plethysmography. Ventilatory responses to hypoxia were significantly weaker in ECE-1+/- than in ECE-1+/+ newborn mice (percentage ventilation increase: 1 +/- 25% versus 33 +/- 29%, p = 0.010). Baseline breathing variables and ventilatory responses to hypercapnia were normal in the ECE-1+/- newborn mice. No differences were observed between adult ECE-1+/- and ECE-1+/+ mice. We conclude that ECE-1 is required for normal ventilatory response to hypoxia at birth.

Arousal response to hypoxia in newborn mice.

We examined the arousal response to 5% O(2) in newborn mice at several ages before and after peripheral chemoreceptor resetting, namely, at 3, 12, and 48 h (n=22 in each group). Breathing was measured by whole-body flow barometric plethysmography. Sleep and arousal were determined behaviourally. We found that: (1) the arousal response was present in all age groups; (2) the arousal response occurred during the hypoxic ventilatory decline in all age groups, showing that mechanoreceptor input was not sufficient to trigger arousal; and (3) arousal latency was shorter after than before chemoreceptor resetting, suggesting a contribution of chemoreceptors to arousal. We conclude that arousal may contribute to the hypoxic ventilatory response in the early postnatal period in mice and that it should be taken into consideration in studies of ventilatory control maturation in newborns.

Ventilatory responses to hypercapnia and hypoxia in Mash-1 heterozygous newborn and adult mice.

Normal control of breathing is characterized by maintenance of CO2 and O2 arterial pressures at constant levels by appropriate ventilatory responses to changes in CO2 production and O2 consumption. Abnormal development of this regulatory system during embryogenesis may produce early impairments in chemosensitivity, as in congenital central hypoventilation syndrome. The present study addresses the role of the mammalian achaetescute homologous gene (Mash-1) in the development of respiratory control. We analyzed ventilatory responses to hypercapnia (8% CO2, 21% O2, 71% N2) and hypoxia (10% O2, 3% CO2, 87% N2) in newborn and adult Mash-1 heterozygous mice (Mash-1+/-) and their wild-type littermates (Mash-1+/+). Ventilation, breath duration, and tidal volume were measured using whole-body plethysmography. Ventilatory responses to hypercapnia were significantly weaker in newborn male Mash-1+/- compared with Mash-1+/+ mice as a result of a weaker breath-duration response. No differences were observed between adult Mash-1+/- and Mash-1+/+ mice. Our data suggest that Mash-1 may be involved in respiratory control development via mechanisms linked to the X chromosome.

Behavioural correlates of conditioned ventilatory responses to hypoxia in rats.

To examine the possible contribution of behavioural arousal to ventilatory conditioning, we performed a differential conditioning experiment using two odours as the paired conditioned stimulus (CS + ) and unpaired conditioned stimulus (CS-) and a hypoxic mixture (7.5% O2) as the unconditioned stimulus (US) in 24 adult male rats. Vanillin was the CS + and rose the CS - in half the rats, and vice versa in the other half. Each rat underwent 26 paired CS + /hypoxia trials and 26 CS - trials in alternation, followed by two CS + only and two CS - trials to test for conditioning. Analysis of breathing variables and behavioural scores during the test showed two qualitatively different conditioned responses. The initial conditioned response was characterised by short breath durations (TT), frequent sniffing episodes, and arousal responses. Following this, a specific, conditioned increase in tidal volume (VT) and levelling off of sniffing and motor activities occurred. The early TT-response and late VT-response to CS + both contributed to an increase in ventilation (VI). The present data show that the association of an odour and hypoxia elicits a biphasic ventilatory conditioned response, of which the first component is integrated into conditioned arousal.

Reliability of a new device to assess the oxygen consumption of human respiratory muscles.

PURPOSE: This study tests the reliability of a new device for assessing the oxygen consumption of the respiratory muscles (VO2 resp.). METHODS: Fourteen healthy male volunteers participated in the study. The device consists of an expandable external ventilatory dead space created with pieces of plastic tubing and a spirometer filled with 100% oxygen. It also incorporates a carbon dioxide absorber. Total VO2 (VO2 tot.) was recorded from the spirometric closed circuit and ventilation (V(E)), from the spirometer tracing. For each subject the test procedure was carried out in duplicate (T1 and T2) after an overnight fast. The dead space was increased at a constant rate of 260 mL every 90 s, and VO2 tot. and V(E) increased progressively. Because log VO2 tot. was linearly related to V(E), we calculated the slope value (log VO2-V(E)) and the Y-intercept (VE = 0) of the semilog regression representing, respectively, VO2 resp. and metabolic VO2 (VO2 met.). RESULTS: When compared with values in the literature, these values did not differ from those recorded in subjects of a similar age group. The VO2 resp. and VO2 met. calculated in T1 and T2 were not different (VO2 resp. = 0.0066 +/- 0.0005 for T1 vs 0.0067 +/- 0.0005 log mL x min(-1)/L x min(-1) for T2 and VO2 met. = 269.3 +/- 28.6 for T1 vs 281.9 +/- 24.1 mL x min(-1) for T2). The coefficients of variation were: 25% at T1 and 23% at T2 for VO2 resp. and 34% at T1 and 29% at T2 for VO2 met. Moreover, significant correlations (r = 0.96, P < 0.001 for VO2 resp., r = 0.95, P < 0.001 for VO2 met.), high coefficients of determination (r2 = 0.92 for VO2 resp., r2 = 0.90 for VO2 met.) and negligible SEE (0.0005 for VO2 resp., 0.2 mL x min(-1) for VO2 met.) were found between the two tests. When we plotted the mean values of VO2 resp. and VO2 met. measured at T1 and T2 against their respective differences, more than 95% of the slight differences ranged between the limits defined by mean value +/- 2 SD, reflecting the small discrepancy between duplicate measurements. CONCLUSION: The results confirm that the test performed with this device is useful and reliable for assessing the VO2 resp. in healthy subjects.

Effects of voluntary changes in breathing frequency on respiratory comfort.

Previous experiments on voluntary breathing have suggested that spontaneous breathing is partly determined by the minimization of respiratory sensations. However, during instructed breathing, respiratory sensations may be confounded with difficulty in achieving the prescribed pattern. In the present experiment, we tested the hypothesis that the subjective assessment of respiratory comfort and the difficulty in following breathing instructions are closely related. A total of 15 subjects adjusted breathing frequency to prescribed values ranging from 40 to 250% of individual spontaneous levels. Then, they scored the difficulty of this task and the discomfort associated with the target frequency. Difficulty scores sharply increased above 100% (spontaneous level) and discomfort scores displayed a similar shape. A significant positive correlation between discomfort and difficulty was found, thus suggesting a possible influence of the difficulty to follow ventilatory instructions on respiratory sensation scores.

The effects of restraint on ventilatory responses to hypercapnia and hypoxia in adult mice.

The aim of this experiment was to determine whether ventilatory measurements in adult restrained mice provide a valid assessment of chemosensitivity. We used whole-body plethysmography to compare breathing patterns in eight restrained and eight unrestrained outbred Swiss mice during air breathing, hypercapnia, and hypoxia. The mice in the restrained group were each placed in a loosely restraining wire-mesh cage. The unrestrained mice could move freely inside the plethysmograph. All the mice received three hypercapnic stimuli (8.5% CO2) and three hypoxic isocapnic stimuli (10% O2, 3.5% CO2). As compared to unrestrained mice, restrained mice had significantly lower breath durations (TT, 445+/-110 ms vs. 323+/-32 ms) and higher ventilation (VE) levels (15.7+/-2.6 microl/(sec x g) vs. 22.2+/-4.5 microl/(sec x g)), whereas no difference was observed for tidal volume (VT). The increases in frequency and ventilation from baseline to hypercapnia were not significantly different in restrained and unrestrained mice. The VE response to hypoxia was marginally higher in restrained mice. We conclude that chemosensitivity to hypercapnia, and to a lesser extent to hypoxia, can be measured in restrained adult mice, but that the baseline breathing pattern cannot.

The effect of attentional load on the breathing pattern in children.

Experiments designed to establish the effects of video games on breathing patterns have led to contradictory results. Several authors reported that video games tended to increase breathing frequency (i.e. to reduce breath duration), whereas others reported the opposite. We postulated that video games contain different psychophysiological components which may have opposite effects on breathing pattern. On the one hand, arousal and emotion may tend to stimulate breathing. On the other, focusing attention on the game may prompt subject to inhibit any movement--including breathing--which might be a potential nuisance variable. The aim of this study was to assess the specific effects of the attentional load in an experimental environment characterized by its low emotional impact. We measured breathing variables, cardiac frequency and cortisol levels in 10 healthy children (mean age = 9.2 +/- 1.5 years) who were familiar with the environment, the experimenter and the video game. Breath duration rose significantly, from 2.56 to 3.16 s, as a function of game difficulty. Cortisol levels, heart rate and the thoracic contribution to breathing displayed no significant changes. Taken together, these data suggest that focusing attention on the game tended to inhibit breathing and that previous contradictory reports in this respect were due to the confounding effects of emotion.

Classical conditioning to hypoxia using odors as conditioned stimuli in rats.

The authors performed a differential conditioning experiment in 30 rats, using 2 odors as the conditioned stimuli (CS+ and CS-) and hypoxia (8% O2) as the unconditioned stimulus. Vanillin was the CS+ and rose the CS- in half of the rats, and vice versa in the other half. Fifteen paired CS+/hypoxia trials and 15 CS- only trials were performed in random order, followed by 3 CS+ only and 3 CS- only trials to test for conditioning. The increase in ventilation from prestimulus levels averaged 116 +/- 85% in response to CS+ versus 55 +/- 36% in response to CS-. This effect was supported by the significant Pre-Post Stimulus x CS Type interaction for this variable (p < .003). The data confirm the sensitivity of breathing to conditioning processes and also indirectly support the hypothesis that feedforward responses may complement feedback reflex pathways in respiratory homeostasis.

Classic conditioning of the ventilatory responses in rats.

Recent authors have stressed the role of conditioning in the control of breathing, but experimental evidence of this role is still sparse and contradictory. To establish that classic conditioning of the ventilatory responses can occur in rats, we performed a controlled experiment in which a 1-min tone [conditioned stimulus (CS)] was paired with a hypercapnic stimulus [8.5% CO2, unconditioned stimulus (US)]. The experimental group (n = 9) received five paired CS-US presentations, followed by one CS alone to test conditioning. This sequence was repeated six times. The control group (n = 7) received the same number of CS and US, but each US was delivered 3 min after the CS. We observed that after the CS alone, breath duration was significantly longer in the experimental than in the control group and mean ventilation was significantly lower, thus showing inhibitory conditioning. This conditioning may have resulted from the association between the CS and the inhibitory and aversive effects of CO2. The present results confirmed the high sensitivity of the respiratory controller to conditioning processes.

Influence of thoracoabdominal pattern of breathing on respiratory resistance.

The purpose of this study was to test the hypothesis that voluntary changes in thoracoabdominal pattern of breathing may increase total respiratory resistance. Thirty-one normal subjects were asked to control their thoracoabdominal pattern of breathing by using a visual feedback. Thoracic and abdominal volume changes were measured by inductance plethysmography. Respiratory resistance and elastance were measured by forced oscillometry. The mean (+/-SD) percent thoracic contributions to tidal volume during thoracic or abdominal breathing were 75 (+/-11) and 25% (+/-9), respectively. These changes induced small but significant increases in resistance (P < 0.005) and elastance (P < 0.002). The increased resistance was observed in 22 subjects for thoracic breathing (P < 0.016) and in 21 subjects for abdominal breathing (P < 0.043). The mean value (+/-SD) of individual increases in resistance during thoracic or abdominal breathing, compared with normal breathing, were 9.2 +/- 17.5 and 9.4 +/- 19.9%, respectively. The fact that departing from spontaneous pattern increases respiratory resistance is consistent with the notion that breathing pattern is optimally adjusted on the basis of mechanical criteria.

Ventilatory responses to imagined exercise.

We studied whether the ventilatory responses to imagined exercise are influenced by automatic processes. Twentynine athletes produced mental images of a sport event with successive focus on the environment, the preparation, and the exercise. Mean breathing frequency increased from 15 to 22 breaths/min. Five participants reported having voluntarily controlled breathing, two of them during preparation. Twenty participants reported that their breathing pattern changed during the experiment: 11 participants were unable to correctly report on the direction of changes in frequency, and 13 incorrectly reported changes in amplitude. This finding suggests that these changes were not voluntary in most participants and may therefore reveal automatic forebrain influences on exercise hyperpnea. However, these changes may also reflect nonspecific processes (e.g., arousal) different from those occurring during actual exercise.

Thoracoabdominal pattern of breathing in neuromuscular disorders.

STUDY OBJECTIVE: To assess abnormalities in thoracoabdominal pattern of breathing (TAPB) in neuromuscular disorders during spontaneous breathing, intermittent positive pressure ventilation (IPPV) with and without abdominal (AB) binder, and immediately after IPPV. DESIGN: Repeated measures design: Pre-IPPV spontaneous breathing, IPPV, IPPV with AB binder, and post-IPPV spontaneous breathing. In protocol 1, ventilator pressure was held constant at the individual value habitually adopted in sessions of IPPV. In protocol 2, it was increased stepwise from 5 to 30 cm H2O. SETTING: University hospital, Department of Pediatrics, Intensive Care, and Neuro-Ventilatory Rehabilitation. PATIENTS: Thirty-one patients with spinal muscular atrophy (SMA) and 19 patients with myopathy, mean age (+/- SD) 9.7 +/- 3 years. MEASUREMENTS: Tidal volume (VT), percent thoracic contribution to VT (%RC), the phase angle between the thoracic and the AB volume changes and the labored breathing index, which is an index of asynchrony taking into account both the phase relationships and relative volumes of rib cage and AB compartments. RESULTS: We observed marked abnormalities in TAPB during spontaneous breathing, especially in the SMA group. %RC, labored breathing index, and phase angle displayed nearly normal values during IPPV. IPPV pressures of 25 to 30 cm H2O were necessary to increase %RC above 80%. AB binding decreased VT, but led to larger thoracic volumes, especially in patients with SMA. Thoracic contribution to VT and thoracic volume after IPPV were higher than baseline levels. CONCLUSIONS: The quantitative assessment of TAPB enhances the ability to estimate pulmonary function in neuromuscular disorders, and the efficiency of mechanical ventilation.