Recurrent hypoxia in a rat model of sleep apnea during pregnancy leads to microglia-dependent respiratory deficits and persistent neuroinflammation in adult male offspring
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These authors provide compelling evidence that gestational intermittent hypoxia, a component of sleep apnea during pregnancy, increases inflammation in the spinal cords of male mice. Increased inflammation is robustly linked to deficits in respiratory plasticity both biochemically and via functional depletion assays. These data are important given the fact that male infants have worse outcomes in the NICU and are at higher risk of sudden infant death syndrome.
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Abstract
Sleep apnea (SA) during pregnancy is detrimental to the health of the pregnancy and neonate, but little is known regarding long-lasting consequences of maternal SA during pregnancy on adult offspring. SA is characterized by repeated cessations in breathing during sleep, resulting in intermittent hypoxia (IH). We show that gestational IH (GIH) in rats reprograms the male fetal neuroimmune system toward enhanced inflammation in a region- and sex-specific manner, which persists into adulthood. Male GIH offspring also had deficits in the neural control of breathing, specifically in the ability to mount compensatory responses to central apnea, an effect that was rescued by a localized anti-inflammatory or microglial depletion. Female GIH offspring appeared unaffected. These results indicate that SA during pregnancy sex- and region-dependently skews offspring microglia toward a pro-inflammatory phenotype, which leads to long-lasting deficits in the capacity to elicit important forms of respiratory neuroplasticity in response to breathing instability. These studies contribute to the growing body of recent evidence indicating that SA during pregnancy may lead to sex-specific neurological deficits in offspring that persist into adulthood.
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eLife assessment
These authors provide compelling evidence that gestational intermittent hypoxia, a component of sleep apnea during pregnancy, increases inflammation in the spinal cords of male mice. Increased inflammation is robustly linked to deficits in respiratory plasticity both biochemically and via functional depletion assays. These data are important given the fact that male infants have worse outcomes in the NICU and are at higher risk of sudden infant death syndrome.
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Reviewer #1 (Public Review):
These authors use a mouse model of gestational intermittent hypoxia (GIH), a component of sleep apnea during pregnancy, to test the hypothesis that GIH induces inflammation in the central nervous system that impairs respiratory functions, in a sex-dependent manner. The major finding of this work is that spinal cord inflammation, mainly driven by activated microglia cells, impairs inactivity-induced inspiratory motor facilitation (iMF). The authors successfully test this hypothesis and their results support their conclusion.
Major strengths of this work include a robust study design, a well-defined translational model (GIH that sets on later in pregnancy), complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies, and sufficient power to …
Reviewer #1 (Public Review):
These authors use a mouse model of gestational intermittent hypoxia (GIH), a component of sleep apnea during pregnancy, to test the hypothesis that GIH induces inflammation in the central nervous system that impairs respiratory functions, in a sex-dependent manner. The major finding of this work is that spinal cord inflammation, mainly driven by activated microglia cells, impairs inactivity-induced inspiratory motor facilitation (iMF). The authors successfully test this hypothesis and their results support their conclusion.
Major strengths of this work include a robust study design, a well-defined translational model (GIH that sets on later in pregnancy), complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies, and sufficient power to evaluate sex differences. In particular, the authors note the upregulation of several NF-kB regulates genes and increased concentration expression of inflammatory markers in the spinal cords of male mice. These mice also have deficits in the iMF response. By depleting microglia and blocking Ik-kinases, the authors convincingly demonstrate that the increased spinal inflammation is causative in the disruption of respiratory plasticity.
The major limitation as the manuscript is currently written is a clear rationale for evaluating the iMF response as a primary endpoint. One of the corresponding authors is an expert in iMF, but there is no rationale for why it is expected that this aspect of plasticity might be disrupted. The authors discuss breathing and respiratory function in the introduction, but these have not been measured here. It is not known whether GIH impacts respiratory response or baseline breathing in a spontaneous breathing model, including baseline frequency and tidal volume and the ventilatory responses to hypoxia and hypercapnia. Shortening the introduction to offer a clear rationale would be beneficial, given the wide audience of this journal. The limitations of this model, including vagotomy, mechanical ventilation, hyperoxic ventilation, and recording from the phrenic nerve in lieu of respiratory measures, should also be discussed in the discussion for readers not familiar with this model outside of the respiratory control field.
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Reviewer #2 (Public Review):
Experiments were designed to determine if the adult offspring of mothers exposed to intermittent hypoxia (IH) during late gestation show reduced compensatory respiratory motor neuron plasticity, which is defined as an increase in respiratory motor system output that persists for a long-time following cessation of the perturbing stimulus. Here, the team uses a clever approach to evoke plasticity, which they term inactivity-induced respiratory motor facilitation. This approach has been shown to be repeatable and robust, and therefore useful for evaluating the impact of experimental interventions on compensatory respiratory motor system responses. The model is a paralyzed, mechanically ventilated, anesthetized rat in which the activity of a phrenic nerve is used as an index of excitability of the phrenic motor …
Reviewer #2 (Public Review):
Experiments were designed to determine if the adult offspring of mothers exposed to intermittent hypoxia (IH) during late gestation show reduced compensatory respiratory motor neuron plasticity, which is defined as an increase in respiratory motor system output that persists for a long-time following cessation of the perturbing stimulus. Here, the team uses a clever approach to evoke plasticity, which they term inactivity-induced respiratory motor facilitation. This approach has been shown to be repeatable and robust, and therefore useful for evaluating the impact of experimental interventions on compensatory respiratory motor system responses. The model is a paralyzed, mechanically ventilated, anesthetized rat in which the activity of a phrenic nerve is used as an index of excitability of the phrenic motor neuron population, which drives the diaphragm muscle in mammals. Importantly, the activity of the respiratory control system in the brainstem can be terminated by reducing the pH of the blood and cerebrospinal fluid (CSF) to a value that is unique to each animal. This value is called the central apneic threshold, and it occurs because pH-sensitive receptors in the brainstem provide critical excitatory synaptic input to the respiratory controller. Since the pH of the blood and CSF depends importantly on the corresponding levels of CO2 the pH can be adjusted up or down by manipulating the blood CO2. To evoke inactivity-induced respiratory motor facilitation, the group first sets the mechanical ventilator at a rate sufficient to reduce CO2 below the apneic threshold to stop phrenic motor output and then keeps the ventilator output at this level. Then, CO2 is added to the ventilator to raise the blood CO2 to levels just above the apneic threshold, which establishes the baseline level of phrenic motor neuron output. They then periodically stop adding CO2 to the inspired gas mixture, which allows CO2 to fall below the apneic threshold, which abolishes phrenic nerve activity. After 1 minute of apnea, the CO2 is reintroduced, blood CO2 levels rise and phrenic nerve activity resumes. This sequence of 1 minute of central apnea followed by 5 minutes of phrenic motor activity is repeated 5 times, and the recording continues for 60 minutes after the fifth apneic episode. As shown in figure 1, a progressive and long-lasting increase in phrenic nerve activity is observed in both male and female control animals, consistent with compensatory respiratory neuroplasticity. Interestingly, the neuroplastic response in the male offspring of animals exposed to intermittent hypoxia throughout gestation was abolished but was unchanged in the female offspring.
This striking, sex-dependent loss of respiratory motor neuroplasticity in the offspring of IH-exposed mothers was associated with increased inflammatory response in the cervical spinal cord, but not in the brainstem. In addition, the transcriptomes of both the spinal cord and brainstems from male offspring of IH-exposed mothers differed from control, with upregulation of genes targeting transcription factors involved in the inflammatory response, specifically the NF-kB/STAT pathways. Accordingly, additional experiments were done to demonstrate that blocking STAT transcription factor activation with intrathecally-delivered drugs restored the plastic response in the male offspring of IH-exposed mothers.
These are novel and interesting observations showing that GIH is associated with a strong, microglia-mediated inflammatory response in the spinal cord of adult males, but not female offspring. The inflammatory response was associated with a loss of compensatory neuroplasticity in phrenic motoneurons. The techniques employed include difficult and labor-intensive whole animal physiology experiments to RNA sequencing and microglial functional analyses. These data are thus important and of wide interest, as they link a gestational insult with spinal cord inflammation, microglial dysfunction, and a sex-dependent alteration in the ability to generate neuromotor plasticity that persists into adulthood. The main caveat is that IH does not model either obstructive or central apnea as both are associated with combined episodic hypoxia and hypercapnia. Moreover, whereas excitatory synaptic input to the phrenic motoneurons was periodically silenced to evoke "inactivity", patients with upper airway obstruction during sleep take great breathing efforts. The model used here seems more like central apnea; do pregnant humans typically have central or obstructive sleep apnea? Nonetheless, the experiments provide important insight into the impact of gestational hypoxia on the development of breathing control in male offspring.
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Reviewer #3 (Public Review):
The role of maternal sleep apnea on neurological and physiological function in the offspring is of substantial interest and the investigators have contributed significantly to this emerging field via prior publications. Recent work has evidenced that recurrent bouts of gestational intermittent hypoxia (GIH) result in life-long changes in cardiovascular, cognitive, and metabolic function in the offspring. Recently, investigators have shown that GIH reprograms the neuroinflammatory response of neonates, such that the newborn offspring's normal immune response is attenuated following a Lipopolysaccharides (LPS) exposure and respiratory rhythm generation is considerably altered (Johnson et al. Respir Physiol Neurobiol. 2018). The present study by Mickelson et al. substantially extends these previous findings by …
Reviewer #3 (Public Review):
The role of maternal sleep apnea on neurological and physiological function in the offspring is of substantial interest and the investigators have contributed significantly to this emerging field via prior publications. Recent work has evidenced that recurrent bouts of gestational intermittent hypoxia (GIH) result in life-long changes in cardiovascular, cognitive, and metabolic function in the offspring. Recently, investigators have shown that GIH reprograms the neuroinflammatory response of neonates, such that the newborn offspring's normal immune response is attenuated following a Lipopolysaccharides (LPS) exposure and respiratory rhythm generation is considerably altered (Johnson et al. Respir Physiol Neurobiol. 2018). The present study by Mickelson et al. substantially extends these previous findings by showing that GIH results in region and sex-specific changes in the microglial activation of adult rats. In male rats, these changes are indicative of an increased pro-inflammatory profile and contribute to the blunted ability to elicit respiratory neuroplasticity following apneic challenge-induced breathing instability. While a robust attenuation of key inflammation-related genes was observed in spinal and brainstem regions of GIH-exposed female rats, these results were not pursued further and present another exciting area of investigation. Nonetheless, the primary goal of these studies was to elucidate the potential role of spinal microglial activation in decreasing respiratory neuroplasticity in adult rats, which has been investigated in-depth using clever and appropriate experimental approaches.
The respiratory motor system employs homeostatic neuroplastic mechanisms at the spinal level to increase phrenic motor output in response to reduced neural activation of respiratory pathways (also called inactivity-induced inspiratory motor facilitation (iMF)). Under carefully controlled conditions, lowering inspired CO2 levels causes cessation of phrenic inspiratory output (central apnea). The authors have previously utilized a protocol of recurrent central apneas to elicit iMF in phrenic motor output. In the present study, authors utilize this neurophysiological outcome to test the impact of GIH on altering the neuroplastic capacity of adult rats. A key finding of this study is that GIH attenuates iMF in male rats. This attenuation is not observed in female rats. To test the role of inflammation (in particular microglia-driven inflammation), the authors employ two approaches to inactivate spinal inflammatory pathways or deplete microglia in adult male rats. Building upon the 29 out of 12982 differentially expressed genes in cervical spinal cord microglia in GIH vs GNX (control exposure rats), the authors targeted the NF-κB pathway using intrathecally delivered TPCA-1 (NF-κB inhibitory subunit (IκB) inhibitor). Indeed, spinal TPCA-1 application restored iMF in GIH-exposed male rats. The second approach employed global microglial depletion using an orally delivered CSF1R inhibitor Pexidartinib (PLX3397) to show that iMF could be provoked in GIH-exposed male rats. It is important to note that although the authors do not report changes in microglial expression in GIH vs. GNX rats, they conclude that there are alterations in microglial activation that contribute to the GIH-induced attenuation of the neuroplastic capacity of respiratory motor networks.
A few questions emerge from this study. In the previous study by the group investigating changes in the inflammatory profile of newborns exposed to GIH, Cox-2 mRNA expression was shown to be elevated in the spinal cords of male rats. This is an interesting finding that has not been tested in GIH-exposed adult male rats in this study and it would be interesting to follow up on whether these changes in microglial profiles are conserved from newborn to adult stages. Indeed, the authors identify additional changes in hypoxia-responsive signaling pathways of GIH rats whose role in impaired respiratory plasticity would be an exciting follow-up to the current study.
The authors emphasize that the reduction in iMF capacity is due to changes in local spinal microglia activation. They do also report that 4 genes were upregulated in the brainstem region of GIH rats as compared to GNX rats. Without an appropriate anatomical control (such as hypoglossal motor output), it would not be appropriate to conclude that microglial activation resulting from GIH has no impact on respiratory networks. Further, the inclusion of bursting frequency data could provide some insight into neural drive originating in brainstem regions.
In summary, this study by Mickelson et al. provides a valuable framework for mechanisms imposing long-lasting changes in respiratory motor control following gestational exposure to episodes of sleep apnea. Furthermore, the work completed here may very well be relevant to other motor systems in which spinal microglia modulate the capacity to elicit homeostatic plastic changes. These changes are particularly important in the context of disease and injury and may impair the capacity of GIH-exposed individuals to elicit neuroplastic changes at the motor neuron level.
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