Erythropoietin-Mediated Regulation of Central Respiratory Command.

Erythropoietin (Epo) is a cytokine expressed throughout the body, including in the central nervous system where it can act as a breathing modulator in the central respiratory network. In vitro, Epo allows maintaining the activity of respiratory neurons during acute hypoxia, resulting in inhibition of the hypoxia-induced rhythm depression. In vivo, Epo action on the central respiratory command results in enhancement of the acute hypoxic ventilatory response, allowing a better oxygenation of the body by improvement of gases exchanges in the lungs. Importantly, this effect of Epo is age-dependent, being observed at adulthood and at both early and late postnatal ages, but not at middle postnatal ages, when an important setup of the central respiratory command occurs. Epo regulation of the central respiratory command involves at least two intracellular signaling pathways, PI3K-Akt and MEK-ERK pathways. However, the exact mechanism underlying the action of Epo on the central respiratory control remains to be deciphered, as well as the exact cell types and nuclei involved in this control. Epo-mediated effect on the central respiratory command is regulated by several factors, including hypoxia, sex hormones, and an endogen antagonist. Although more knowledge is needed before reaching the clinical trial step, Epo seems to be a promising therapeutic treatment, notably against newborn breathing disorders.

Consequences of maternal omega-3 polyunsaturated fatty acid supplementation on respiratory function in rat pups.

KEY POINTS: Incomplete development of the neural circuits that control breathing contributes to respiratory disorders in pre-term infants. Manifestations include respiratory instability, prolonged apnoeas and poor ventilatory responses to stimuli. Based on evidence suggesting that omega-3 polyunsaturated fatty acids (n-3 PUFA) improves brain development, we determined whether n-3 PUFA supplementation (via the maternal diet) improves respiratory function in 10-11-day-old rat pups. n-3 PUFA treatment prolonged apnoea duration but augmented the relative pulmonary surface area and the ventilatory response to hypoxia. During hypoxia, the drop in body temperature measured in treated pups was 1 °C less than in controls. n-3 PUFA treatment also reduced microglia cell density in the brainstem. Although heterogeneous, the results obtained in rat pups constitute a proof of concept that n-3 PUFA supplementation can have positive effects on neonatal respiration. This includes a more sustained hypoxic ventilatory response and a decreased respiratory inhibition during laryngeal chemoreflex. ABSTRACT: Most pre-term infants present respiratory instabilities and apnoeas as a result of incomplete development of the neural circuits that control breathing. Because omega-3 polyunsaturated fatty acids (n-3 PUFA) benefit brain development, we hypothesized that n-3 PUFA supplementation (via the maternal diet) improves respiratory function in rat pups. Pups received n-3 PUFA supplementation from an enriched diet (13 g kg-1 of n-3 PUFA) administered to the mother from birth until the experiments were performed (postnatal days 10-11). Controls received a standard diet (0.3 g kg-1 of n-3 PUFA). Breathing was measured in intact pups at rest and during hypoxia (FiO2  = 0.12; 20 min) using whole body plethysmography. The duration of apnoeas induced by stimulating the laryngeal chemoreflex (LCR) was measured under anaesthesia. Lung morphology was compared between groups. Maternal n-3 PUFA supplementation effectively raised n-3 PUFA levels above control levels both in the blood and brainstem of pups. In intact, resting pups, n-3 PUFA increased the frequency and duration of apnoeas, especially in females. During hypoxia, n-3 PUFA supplemented pups hyperventilated 23% more than controls; their anapyrexic response was 1 °C less than controls. In anaesthetized pups, n-3 PUFA shortened the duration of LCR-induced apnoeas by 32%. The relative pulmonary surface area of n-3 PUFA supplemented pups was 12% higher than controls. Although n-3 PUFA supplementation augments apnoeas, there is no clear evidence of deleterious consequences on these pups. Based on the improved lung architecture and responses to respiratory challenges, this neonatal treatment appears to be beneficial to the offspring. However, further experiments are necessary to establish its overall safety.

Oxygen Sensing in Early Life.

From birth, animals should possess functional machinery to appropriately regulate its respiration. This machinery has to detect the available oxygen quantity in order to efficiently modulate breathing movements in accordance with body requirements. The chemosensitivity process responsible for this detection is known to be mainly performed by carotid bodies. However, pulmonary neuroendocrine cells, which are mainly gathered in neuroepithelial bodies, also present the capability to exert chemosensitivity. The goal of this article is to put in perspective the potential complementarity in the activity of these two peripheral chemosensors in the context of neonatal oxygen chemosensitivity.

Induction of serpinb1a by PACAP or NGF is required for PC12 cells survival after serum withdrawal.

PC12 cells are used to study the signaling mechanisms underlying the neurotrophic and neuroprotective activities of pituitary adenylate cyclase-activating polypeptide (PACAP) and nerve growth factor (NGF). Previous microarray experiments indicated that serpinb1a was the most induced gene after 6 h of treatment with PACAP or NGF. This study confirmed that serpinb1a is strongly activated by PACAP and NGF in a time-dependent manner with a maximum induction (~ 50-fold over control) observed after 6 h of treatment. Co-incubation with PACAP and NGF resulted in a synergistic up-regulation of serpinb1a expression (200-fold over control), suggesting that PACAP and NGF act through complementary mechanisms. Consistently, PACAP-induced serpinb1a expression was not blocked by TrkA receptor inhibition. Nevertheless, the stimulation of serpinb1a expression by PACAP and NGF was significantly reduced in the presence of extracellular signal-regulated kinase, calcineurin, protein kinase A, p38, and PI3K inhibitors, indicating that the two trophic factors share some common pathways in the regulation of serpinb1a. Finally, functional investigations conducted with siRNA revealed that serpinb1a is not involved in the effects of PACAP and NGF on PC12 cell neuritogenesis, proliferation or body cell volume but mediates their ability to block caspases 3/7 activity and to promote PC12 cell survival.

Brain-derived neurotrophic factor interacts with astrocytes and neurons to control respiration.

Respiratory rhythm is generated and modulated in the brainstem. Neuronal involvement in respiratory control and rhythmogenesis is now clearly established. However, glial cells have also been shown to modulate the activity of brainstem respiratory groups. Although the potential involvement of other glial cell type(s) cannot be excluded, astrocytes are clearly involved in this modulation. In parallel, brain-derived neurotrophic factor (BDNF) also modulates respiratory rhythm. The currently available data on the respective roles of astrocytes and BDNF in respiratory control and rhythmogenesis lead us to hypothesize that there is BDNF-mediated control of the communication between neurons and astrocytes in the maintenance of a proper neuronal network capable of generating a stable respiratory rhythm. According to this hypothesis, progression of Rett syndrome, an autism spectrum disease with disordered breathing, can be stabilized in mouse models by re-expressing the normal gene pattern in astrocytes or microglia, as well as by stimulating the BDNF signaling pathway. These results illustrate how the signaling mechanisms by which glia exerts its effects in brainstem respiratory groups is of great interest for pathologies associated with neurological respiratory disorders.

Using plethysmography to determine erythropoietin's impact on neural control of ventilation.

The evaluation of respiratory parameters often requires the use of anesthetics (that depress the neural -network controlling respiration), and/or ways to restrain the animal's mobility (that produces a stress-dependent increase of respiration). Consequently, the establishment of plethysmography represented an invaluable technique in respiratory physiology. Plethysmography, indeed, allows the assessment of ventilatory parameters on living, unanesthetized, and unrestrained animals. The conception of the barometric plethysmography relies on the fact that an animal placed inside a hermetically closed chamber generates through its breathing a fluctuation of pressure in the chamber than can be recorded. Thus, the respiratory frequency and the tidal volume can be directly measured, while the animal's ventilation is calculated indirectly by the multiplication of these two parameters. In our hands, plethysmography was a key tool to investigate the impact of erythropoietin (Epo) on the neural control of hypoxic ventilation in mice.

Erythropoietin and the sex-dimorphic chemoreflex pathway.

During hypoxic or hypoxemic conditions, tissue oxygenation and arterial O(2) carrying capacity are upregulated by two complementary systems, namely the neural respiratory network (central and peripheral) that leads to increased minute ventilation thereby increasing tissue oxygenation, and erythropoietin (Epo) release by the kidney that activates erythropoiesis in bone marrow to augment arterial blood O(2) carrying capacity. Despite the fact that both neural respiratory control and Epo-mediated elevation of red blood cells are responsible for keeping arterial O(2) content optimal, no interaction between these systems has been described so far. Here we review data obtained in our laboratory demonstrating that ventilatory and erythropoietic systems are tightly connected. We found Epo is the key factor mediating this relationship through modulation of the chemoreflex pathway. Moreover, we showed that this interaction occurs in a sex-dependent manner.

Erythropoietin and its antagonist regulate hypoxic fictive breathing in newborn mice.

Clinical use of erythropoietin in adult and newborn patients has revealed its involvement in neuroprotection, neurogenesis, and angiogenesis. More recently, we showed in adult mouse, that brain erythropoietin interacts with the major brainstem centers associated with respiration to enhance the ventilatory response to acute and chronic conditions of physiological hypoxia (e.g., as occurring at high altitude). However, whether brain erythropoietin is involved in breathing regulation in newborns remains unknown. In this study, en bloc brainstem-spinal cord preparations were obtained from mice at postnatal day 4. After various periods (30, 60, or 90 min) of incubation with 0, 25, or 250 U of erythropoietin, preparations were superfused with artificial cerebrospinal fluid bubbled with normoxic or hypoxic gas mixtures. The electrophysiological fictive breathing produced by axons at the C4 ventral root was next recorded. Our results show that erythropoietin attenuates the hypoxia-mediated decrease of the central respiratory activity and improves post-hypoxic recovery. Additional analysis revealed that the soluble erythropoietin receptor (the endogenous erythropoietin antagonist) dramatically decreases neural hypoxic respiratory activity, confirming the specific erythropoietin effect on respiratory drive. These results imply that erythropoietin exerts main modulation and maintenance of respiratory motor output during hypoxic and post-hypoxic challenges in 4-days old mice.

Life-long consequences of postnatal normoxia exposure in rats raised at high altitude.

We tested the hypothesis that exposure of high-altitude (HA) rats to a period of postnatal normoxia has long-term consequences on the ventilatory and hematological acclimatization in adults. Male and female HA rats (3,600 m, Po(2) ≃ 100 Torr; La Paz, Bolivia) were exposed to normal room air [HA control (HACont)] or enriched oxygen (32% O(2); Po(2) ≃ 160 Torr) from 1 day before to 15 days after birth [HA postnatal normoxia (HApNorm)]. Hematocrit and hemoglobin values were assessed at 2, 12, and 32 wk of age. Cardiac and lung morphology were assessed at 12 wk by measuring right ventricular hypertrophy (pulmonary hypertension index) and lung air space-to-tissue ratio (indicative of alveolarization). Respiratory parameters under baseline conditions and in response to 32% O(2) for 10 min (relieving the ambient hypoxic stimulus) were measured by whole body plethysmography at 12 wk. Finally, we performed a survival analysis up to 600 days of age. Compared with HACont, HApNorm rats had reduced hematocrit and hemoglobin levels at all ages (both sexes); reduced right ventricular hypertrophy (both sexes); lower air space-to-tissue ratio in the lungs (males only); reduced CO(2) production rate, but higher oxygen uptake (males only); and similar respiratory frequency, tidal volume, and minute ventilation. When breathing 32% O(2), HApNorm male rats had a stronger decrease of minute ventilation than HACont. HApNorm rats had a marked tendency toward longer survival throughout the study. We conclude that exposure to ambient hypoxia during postnatal development in HA rats has deleterious consequences on acclimatization to hypoxia as adults.

Enhanced erythropoietin expression in the brainstem of newborn rats at high altitude.

In addition to its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions against acute and delayed degenerative diseases of the brain. Moreover, we have recently demonstrated that endogenously synthesized Epo and soluble Epo receptor (a negative regulator of Epo binding to the Epo receptor) in the central nervous system play a crucial role in facilitating the ventilatory response and acclimatization to hypoxia. Here we hypothesized that cerebral Epo in the brainstem is implicated in the process that allows cardiorespiratory acclimatization to high altitude hypoxia during the postnatal period. Thus, we evaluated the postnatal ontogeny of cerebral Epo concentration of Sprague-Dawley rats living and reproducing at high altitude for longer than 19 years (3600 m in La Paz, Bolivia). Our results show that postnatal Epo concentration in high-altitude rats is higher in the brainstem than in the forebrain. Moreover, although Epo concentration in the forebrain of high-altitude rats is similar to sea-level controls, Epo level in the brainstem is surprisingly 2-fold higher in high-altitude rats than in sea-level controls. These findings strongly suggest that brainstem Epo plays an important role in tolerance to high altitude hypoxia after birth. From a clinical perspective, a better understanding of the role of Epo in the postnatal development of cardiorespiratory responses in neonates exposed to acute or chronic hypoxia might be useful.

Promoting the "3Rs" principle in developmental biology with early and convenient diagnosis of pregnancy in mice.

The 3Rs stand for Replacement of animals in experiments, Reduction in the number of experimental animals, and Refinement of experiments to minimize animal pain and stress. We propose to address Reduction and Refinement in the use of mice as experimental models in developmental research. This study focuses on the maternal percentage of weight increase at gestational day 8 (%WI(GD8)) to diagnose pregnancy early in BALB/c mice. We documented sensitivity, specificity, false positive and negative rates and probability of pregnancy associated with %WI(GD8). This predictive model of pregnancy allows for significant reduction in the number of mice to be sacrificed in developmental research. Reported observations and literature suggest that this model is independent of litter size and should be applicable to other mice strains. This procedure allows mice pregnancy detection before midgestation and proposes an ethically sound approach to experimental animal use by optimizing the number of mice used and refining animal manipulation.

PACAP and a novel stable analog protect rat brain from ischemia: Insight into the mechanisms of action.

Pituitary adenylate cyclase-activating polypeptide (PACAP) shows potent protective effects in numerous models of neurological insults. However, the use of PACAP as a clinically efficient drug is limited by its poor metabolic stability. By combining identification of enzymatic cleavage sites with targeted chemical modifications, a metabolically stable and potent PACAP38 analog was recently developed. The neuroprotective activity of this novel compound was for the first time evaluated and compared to the native peptide using a rat model of middle cerebral artery occlusion (MCAO). Our results show that as low as picomolar doses of PACAP38 and its analog strongly reduce infarct volume and improve neurological impairment induced by stroke. In particular, these peptides inhibit the expression of Bcl-2-associated death promoter, caspase 3, macrophage inflammatory protein-1α, inducible nitric oxide synthase 2, tumor necrosis factor-α mRNAs, and increase extracellular signal-regulated kinase 2, B-cell CLL/lymphoma 2 and interleukin 6 mRNA levels. These results indicate that the neuroprotective effect of PACAP after MCAO is not only due to its ability to inhibit apoptosis but also to modulate the inflammatory response. The present study highlights the potential therapeutic efficacy of very low concentrations of PACAP or its metabolically stable derivative for the treatment of stroke.

Protective effects of pituitary adenylate cyclase-activating polypeptide (PACAP) against apoptosis.

Apoptosis is a regulated process leading to cell death, which is implicated both in normal development and in various pathologies including heart failure, stroke and neurodegenerative diseases. Caspase-3, a key enzyme of the apoptotic pathway, is considered as a major target for the treatment of abnormal cell death. Many factors that inhibit cell death have been identified, but the mechanisms involved are not always fully understood. Pituitary adenylate cylase-activating polypeptide (PACAP) has been shown to exert neuroprotective activities during development. PACAP also inhibits apoptosis in cardiomyopathy, decreases glutamate-induced retinal injury, reduces neuronal loss in case of stroke, and prevents ethanol neurotoxicity. Most of the antiapoptotic effects of PACAP are mediated through the PAC1 receptor. This receptor activates a transduction cascade of second messengers to stimulate Bcl-2 expression which inhibits cytochrome c release and blocks in turn caspase activation. PACAP also acts through the PI3K/Akt pathway and inhibits the expression of proapoptotic factors such as c-Jun or Bax. The remarkable effect of PACAP on the apoptotic cascade suggests that innovative PACAP derivatives could potentially be useful for treatment of post-traumatic lesions, chronic neurodegenerative diseases, cardiac ischemia and/or retinopathy.

Sex hormone metabolism in lung development and maturation.

Sex hormones are increasingly recognized as regulators of lung development. Respiratory distress syndrome (RDS) is the leading cause of morbidity in preterm neonates and occurs with a higher incidence in males. The mechanisms underlying the effects of androgens on lung development and the occurrence of RDS are only partially deciphered, and positive roles of estrogens on surfactant production and alveologenesis are relevant to our understanding of pulmonary diseases. This manuscript reviews current knowledge on androgen and estrogen metabolism and on relevant hormone targets in the fetal lung. Further investigations are needed to elucidate mechanisms orchestrating sex hormone effects on lung development. These studies aim to decrease mortality and morbidity associated with RDS and other pathologies related to lung immaturity at birth.

Involvement of stathmin 1 in the neurotrophic effects of PACAP in PC12 cells.

Rat pheochromocytoma PC12 cells have been widely used to investigate the neurotrophic activities of pituitary adenylate cyclase-activating polypeptide (PACAP). In particular, PACAP has been shown to promote differentiation and to inhibit apoptosis of PC12 cells. In order to identify the mechanisms mediating these effects, we sought for proteins that are phosphorylated upon PACAP treatment. High-performance liquid chromatography and 2D gel electrophoresis analysis, coupled with mass spectrometry, revealed that stathmin 1 is strongly phosphorylated within only 5 min of exposure to PACAP. Western blot experiments confirmed that PACAP induced a robust phosphorylation of stathmin 1 in a time-dependent manner. On the other hand, PACAP decreased stathmin 1 gene expression. Investigations of the signaling mechanisms known to be activated by PACAP revealed that phosphorylation of stathmin 1 was mainly mediated through the protein kinase A and mitogen-activated protein kinase pathways. Blockage of stathmin 1 expression with small interfering RNA did not affect PC12 cell differentiation induced by PACAP but reduced the ability of the peptide to inhibit caspase 3 activity and significantly decreased its neuroprotective action. Taken together, these data demonstrate that stathmin 1 is involved in the neurotrophic effect of PACAP in PC12 cells.

Gene expression profile of androgen modulated genes in the murine fetal developing lung.

BACKGROUND: Accumulating evidences suggest that sex affects lung development. Indeed, a higher incidence of respiratory distress syndrome is observed in male compared to female preterm neonates at comparable developmental stage and experimental studies demonstrated an androgen-related delay in male lung maturation. However, the precise mechanisms underlying these deleterious effects of androgens in lung maturation are only partially understood. METHODS: To build up a better understanding of the effect of androgens on lung development, we analyzed by microarrays the expression of genes showing a sexual difference and those modulated by androgens. Lungs of murine fetuses resulting from a timely mating window of 1 hour were studied at gestational day 17 (GD17) and GD18, corresponding to the period of surge of surfactant production. Using injections of the antiandrogen flutamide to pregnant mice, we hunted for genes in fetal lungs which are transcriptionally modulated by androgens. RESULTS: Results revealed that 1844 genes were expressed with a sexual difference at GD17 and 833 at GD18. Many genes were significantly modulated by flutamide: 1597 at GD17 and 1775 at GD18. Datasets were analyzed by using in silico tools for reconstruction of cellular pathways. Between GD17 and GD18, male lungs showed an intensive transcriptional activity of proliferative pathways along with the onset of lung differentiation. Among the genes showing a sex difference or an antiandrogen modulation of their expression, we specifically identified androgen receptor interacting genes, surfactant related genes in particularly those involved in the pathway leading to phospholipid synthesis, and several genes of lung development regulator pathways. Among these latter, some genes related to Shh, FGF, TGF-beta, BMP, and Wnt signaling are modulated by sex and/or antiandrogen treatment. CONCLUSION: Our results show clearly that there is a real delay in lung maturation between male and female in this period, the latter pursuing already lung maturation while the proper is not yet fully engaged in the differentiation processes at GD17. In addition, this study provides a list of genes which are under the control of androgens within the lung at the moment of surge of surfactant production in murine fetal lung.

Inhibitory effect of PACAP on caspase activity in neuronal apoptosis: a better understanding towards therapeutic applications in neurodegenerative diseases.

Programmed cell death, which is part of the normal development of the central nervous system, is also implicated in various neurodegenerative disorders. Cysteine-dependent aspartate-specific proteases (caspases) play a pivotal role in the cascade of events leading to apoptosis. Many factors that inhibit cell death have now been identified, but the underlying mechanisms are not fully understood. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to exert neurotrophic activities during development and to prevent neuronal apoptosis induced by various insults such as ischemia. Most of the neuroprotective effects of PACAP are mediated through the PAC1 receptor. This receptor activates a transduction cascade of second messengers to stimulate Bcl-2 expression, which inhibits cytochrome c release and blocks the activation of caspases. The inhibitory effect of PACAP on the apoptotic cascade suggests that selective, stable, and potent PACAP derivatives could potentially be of therapeutic value for the treatment of post-traumatic and/or chronic neurodegenerative processes.

Identification of cellular processes that are rapidly modulated in response to tracheal occlusion within mice lungs.

Lung development progresses through a process reliant on mechanical cell stretch. However, this process is not well defined at the molecular level. Our goal was to globally analyze the transcriptome of fetal mouse lungs following short periods of tracheal occlusion (TO) to identify cellular processes that are rapidly modulated in response to intraluminal stretch increase. Serial analysis of gene expression (SAGE) was used to examine the global transcriptomic response of mouse prealveolar stage lungs to in vivo TO at 1 and 3 h. SAGE results were extended by histo- and immunochemical examination. Based on the 97 TO-modulated transcripts identified, our results further point out that continuous stretch in developing lungs leads directly to rapid and highly specific phenotypic modifications in a significant proportion of pulmonary cells. We conclude that intraluminal stretch increase during prealveolar stage of lung development induces a critical transition of pulmonary cells phenotype in which there is an increase in alpha-smooth muscle actin (alpha-SMA)-containing cells along with a relative decrease in lipid-containing cells.

Short-term response to tracheal occlusion during perinatal lung development in mice.

Tracheal occlusion (TO) is known to stimulate lung growth. The aim of this research is to investigate early cellular responses to TO during perinatal growth in order to identify cellular targets in fetal mouse lungs that respond rapidly to surgically induced stretch. TO, or sham-TO, surgery was performed at 16.5 gestational days in fetal mice. Cellular activation, proliferation, and apoptosis were assessed on lung tissue sections harvested at various time points within the first 24 hours after the surgery. Lung tissue from unoperated fetuses and newborn mice served as controls to establish the pattern of cellular proliferation and apoptosis during normal lung development. When compared with sham-TO, TO induces a significantly higher expression of pulmonary c-Fos mRNA within 1 hour after surgery. When compared with sham-TO and unoperated controls, TO induces a rapid (1-hour) increase in proliferating cell nuclear antigen (PCNA) expression within differentiated epithelial airways. In contrast, a significant increase in the apoptotic index of mesenchymal cells from TO lungs was not observed before 24 hours when compared with sham-TO and controls. The data demonstrate that in vivo TO induces an immediate cellular response and that stretch both primarily and significantly accelerates epithelial cell proliferation and mesenchymal cell apoptosis. Moreover, we conclude that TO reduces the gestational time required to reach the natural peaks of proliferation and apoptosis associated with normal lung development, thus resulting in an apparent stimulation of lung growth.

Modulating effect of a selective endothelin A receptor antagonist on pulmonary endothelin system protein expression in experimental diaphragmatic hernia.

BACKGROUND/PURPOSE: Previously, we reported that perinatal administration of atrasentan, a selective endothelin A receptor (ETA) antagonist, provided a beneficial effect on the cardiopulmonary profile under short-term conditions in newborn lambs with surgically induced congenital diaphragmatic hernia (CDH). We hypothesized that changes in the hemodynamic profile that we observed at birth in treated animals could be influenced by pulmonary modulation of the endothelin (ET) system. METHODS: The effect of atrasentan on protein expression levels of ETs and ET receptors (ETA and ETB receptor) was investigated by immunohistochemistry in lung tissues of untreated control (n = 3), treated control (n = 6), untreated CDH (n = 6), and treated CDH newborn lambs (n = 8). RESULTS: Right lung tissue of treated control lambs showed significantly higher ETA protein expression levels in both vascular adventitia and airway epithelia when compared with that of untreated control lambs (P < .05). In contrast, protein expression levels of ETA and ETB receptor were significantly lower in the vascular smooth muscle cells among other tissue subcompartments of the right lung of treated CDH newborn lambs vs CDH lambs (P < .02 and P = .005, respectively). CONCLUSIONS: We speculate that rapid pulmonary modulation of ET system protein expression levels by atrasentan results from an indirect effect possibly dependent on ventilation and/or perfusion. In CDH groups, this could contribute to the beneficial effect of the treatment.