Ventilatory and carotid body responses to acute hypoxia in rats exposed to chronic hypoxia during the first and second postnatal weeks

https://doi.org/10.1016/j.resp.2020.103400Get rights and content

Highlights

  • Rats were exposed to chronic hypoxia (CH) for the first or second postnatal week.

  • CH blunted the hypoxic ventilatory response (HVR) in both age groups.

  • The blunted HVR was associated with diminished carotid body O2 sensitivity.

  • CH during the first (but not second) week delayed maturation of the biphasic HVR.

  • The anti-inflammatory drug ibuprofen did not block this respiratory plasticity.

Abstract

Chronic hypoxia (CH) during postnatal development causes a blunted hypoxic ventilatory response (HVR) in neonatal mammals. The magnitude of the HVR generally increases with age, so CH could blunt the HVR by delaying this process. Accordingly, we predicted that CH would have different effects on the respiratory control of neonatal rats if initiated at birth versus initiated later in postnatal development (i.e., after the HVR has had time to mature). Rats had blunted ventilatory and carotid body responses to hypoxia whether CH (12 % O2) occurred for the first postnatal week (P0 to P7) or second postnatal week (P7 to P14). However, if initiated at P0, CH also caused the HVR to retain the “biphasic” shape characteristic of newborn mammals; CH during the second postnatal week did not result in a biphasic HVR. CH from birth delayed the transition from a biphasic HVR to a sustained HVR until at least P9–11, but the HVR attained a sustained (albeit blunted) phenotype by P13–15. Since delayed maturation of the HVR did not completely explain the blunted HVR, we tested the alternative hypothesis that the blunted HVR was caused by an inflammatory response to CH. Daily administration of the anti-inflammatory drug ibuprofen (4 mg kg−1, i.p.) did not alter the effects of CH on the HVR. Collectively, these data suggest that CH blunts the HVR in neonatal rats by impairing carotid body responses to hypoxia and by delaying (but not preventing) postnatal maturation of the biphasic HVR. The mechanisms underlying this plasticity require further investigation.

Introduction

Chronic exposures to low oxygen levels elicit plasticity in the control of breathing in humans and other mammals, profoundly altering eupneic ventilation and protective ventilatory chemoreflexes (Pamenter and Powell, 2016; Bavis and MacFarlane, 2017). Interestingly, the effects of chronic hypoxia (CH) on some aspects of respiratory control, notably the acute hypoxic ventilatory response (HVR), may be quite different in newborn and adult mammals. In adult mammals, for example, exposures to sustained normobaric or hypobaric hypoxia lasting from several hours to several weeks produce ventilatory acclimatization to hypoxia (VAH), a progressive increase in ventilation associated with an augmented HVR and persistent hyperventilation after acute return to normoxia (Dempsey et al., 2014; Pamenter and Powell, 2016). In contrast, similar durations of hypoxia from birth generally have the opposite effect on the HVR of newborn mammals (Bavis, 2005). Several studies have documented that children born at high altitude (Lahiri et al., 1976; Lahiri, 1981) or who experience hypoxemia secondary to cyanotic heart disease (Sorensen and Severinghaus, 1968; Edelman et al., 1970; Blesa et al., 1977) develop blunted HVR, suggesting that CH attenuates the HVR in newborns rather than enhancing it. This effect has been confirmed in animal experiments in which neonatal rats (Eden and Hanson, 1987a; Wyatt et al., 1995; Reeves and Gozal, 2006; Mayer et al., 2014; MacFarlane et al., 2016; Stryker et al., 2018), cats (Hanson et al., 1989a, b), and sheep (Sladek et al., 1993) exposed to CH (5–30 days in 10–15 % O2, depending on the study) were shown to have reduced HVR compared to individuals reared in normoxia.

Changes in the HVR after CH are attributed (at least in part) to altered function of the carotid bodies, the peripheral arterial chemoreceptors that monitor partial pressure of O2 (Po2) in the arterial blood and initiate the ventilatory response. Whereas CH increases carotid body O2 sensitivity and the gain for the central integration of carotid chemoafferent input in adult mammals (Powell et al., 2000; Teppema and Dahan, 2010; Kumar and Prabhakar, 2012; Pamenter and Powell, 2016), carotid body responsiveness decreases when CH begins at birth. For example, carotid chemoreceptor responses to hypoxia recorded from the carotid sinus nerve (CSN) were reduced in juvenile rats (20–28 d of age; (Donnelly and Doyle, 1994)) and cats (12–23 d of age; (Hanson et al., 1989a)) born and reared in hypoxia. Moreover, glomus (type I) cells isolated from CH-reared neonatal rats exhibited smaller depolarization responses (Wyatt et al., 1995) and intracellular Ca2+ ([Ca2+]i) responses (Sterni et al., 1999) to acute hypoxia, indicating that the diminished afferent nerve activity has a presynaptic origin.

Carotid body chemoreceptors exhibit a marked “resetting” of O2 sensitivity in the early postnatal period, with the threshold Po2 at which carotid body activity increases gradually shifting to higher values to accommodate the greater arterial Po2 in the postnatal environment (Carroll and Kim, 2013). Accordingly, the magnitude of the carotid body and ventilatory responses to hypoxia increase over the first few postnatal days or weeks in newborn mammals (Bissonnette, 2000; Teppema and Dahan, 2010). It has been suggested that the postnatal rise in Po2 is itself a key regulator of carotid body resetting (Carroll and Kim, 2013). Indeed, an earlier rise in Po2 appears to cause premature resetting of carotid body chemoreceptors in fetal lambs (Blanco et al., 1988). It is possible, therefore that decreased carotid body O2 sensitivity (and thus the blunted HVR) observed after chronic postnatal hypoxia represents delayed maturation of the carotid body chemoreceptors (Landauer et al., 1995; Sterni et al., 1999); in other words, by extending the relatively hypoxic in utero environment into the postnatal period, the carotid body resetting process may be slowed or completely prevented. If this hypothesis is correct, one might predict that CH would not attenuate the carotid body or ventilatory responses to hypoxia if the exposure is initiated in the postnatal period but after the carotid chemoreceptors have already reset. We are not aware of any studies that have tested this prediction with respect to carotid body function, although there is evidence that the steady-state HVR is blunted in rats exposed to CH beginning at 11 days of age (Mayer et al., 2014).

Changes to carotid body function may not be solely responsible for abnormal development of the HVR. Newborn mammals exhibit a strongly biphasic HVR in which an initial increase in ventilation is quickly followed by a decline in ventilation toward or below baseline levels. Whereas the initial ventilatory increase (i.e., the early phase) is dominated by the activation of the carotid bodies, the subsequent decline (i.e., the late phase) represents the balance between carotid body stimulation and inhibitory processes within the CNS (Bissonnette, 2000; Teppema and Dahan, 2010). As mammals mature, however, the HVR is characterized by a more sustained increase in ventilation due to diminished CNS inhibition. Like the resetting of carotid chemoreceptor O2 sensitivity, there is some evidence that this transition is modulated by perinatal oxygenation. Specifically, chronic postnatal hyperoxia causes the transition from the biphasic HVR to the sustained HVR to occur earlier in neonatal rats (Bavis et al., 2010, 2014). Similarly, the biphasic HVR may persist longer in rats and cats born into hypoxia (Eden and Hanson, 1987a; Hanson et al., 1989a; Joseph et al., 2000), although the persistence of the biphasic HVR during CH has not been evident in all rat studies (Reeves and Gozal, 2006) nor in sheep (Sladek et al., 1993). Moreover, whether a biphasic HVR would re-emerge if hypoxia is initiated later in the postnatal period (i.e., after already a sustained HVR phenotype) has not been studied.

The cellular and molecular mechanisms linking CH to changes in respiratory control are not fully understood, but recent studies indicate that inflammatory processes influence respiratory plasticity in adult animals exposed to hypoxia. Sustained or intermittent hypoxia can activate microglia in the brainstem and increase the expression of pro-inflammatory molecules in peripheral and central tissues critical to the control of breathing (reviewed in (Hocker et al., 2017; Pena-Ortega, 2019)). In adults, CH-induced inflammation contributes to the peripheral and central mechanisms underlying VAH (Liu et al., 2011b; Hocker et al., 2017). CH increases the numbers of macrophages and the expression of cytokines (IL-6, IL-1β, TNFα) and chemokines (MCP-1) within the carotid body, while administration of anti-inflammatory drugs (ibuprofen, dexamethasone) diminish these effects and block the expected increase in carotid body O2 sensitivity (Liu et al., 2009, 2013). Similarly, chronic administration of ibuprofen prevents the increase in hypoxic ventilation characteristic of VAH in adult rats (Popa et al., 2011) and humans (Basaran et al., 2016). Importantly, while these data point toward excitatory effects of neuroinflammation on respiratory control, inflammatory processes may also have inhibitory effects on respiratory control and respiratory plasticity (e.g., (Herlenius, 2011; Gauda et al., 2013; Hocker et al., 2017)). In neonatal rats, for example, lipopolysaccharide (LPS) exposure elicits a systemic inflammatory response that is associated with abnormal carotid body structure, decreased carotid body responses to hypoxia, and decreased ventilatory responses to changing O2 levels (Master et al., 2016). Moreover, CH in neonatal rats (beginning at 11 days of age) increases the activation of microglia in the brainstem, and this contributes to the blunted HVR observed in these animals (MacFarlane et al., 2016). Therefore, it is possible that CH-induced activation of inflammatory signaling pathways also contribute to the blunted HVR of rats born and raised in hypoxia.

The present study addressed two overarching hypotheses. First, we tested the hypothesis that chronic postnatal hypoxia attenuates the HVR of neonatal rats by delaying the maturation of its peripheral (early phase) and central (late phase) components. If CH delays normal postnatal maturation, we predicted that hypoxia would have different effects on carotid body function and the biphasic HVR if initiated at birth versus initiated at a later postnatal age (i.e., after the carotid body and HVR attain their mature phenotypes). Further, we predicted that prolonging the period of hypoxia from birth would continue to delay the transition from the biphasic HVR to the sustained HVR. Second, we tested the hypothesis that inflammatory signaling processes contribute to the blunted HVR in neonatal rats reared in CH. Specifically, we predicted that chronic ibuprofen treatment would prevent the effects of CH on the acute HVR of neonatal rats.

Section snippets

Methods

All experimental protocols were approved by the Bates College Institutional Animal Care and Use Committee.

Body mass

Rat pups exposed to CH were smaller than age-matched control pups reared in normoxia (Table 1). CH rats exposed for the first postnatal week (P0 to P7) weighed 27 % less than their Control counterparts (P < 0.001). Similar durations of hypoxia during the second postnatal week (P7-P14) reduced body mass as well, but to a lesser extent (approximately 12 % smaller; P < 0.001). Longer exposures to postnatal hypoxia (i.e., 9–15 days beginning at or just prior to birth) reduced the average body mass

Discussion

Neonatal rats exposed to CH had blunted HVR, consistent with previous studies on rats (Eden and Hanson, 1987a; Wyatt et al., 1995; Reeves and Gozal, 2006; Mayer et al., 2014), cats (Hanson et al., 1989a, b), and sheep (Sladek et al., 1993) exposed to hypoxia during development. This effect was evident whether CH was initiated at birth or at 7 days of age, but the proximate mechanisms responsible for the blunting depended on when CH began. In rats exposed to hypoxia from P0 to P7, the decreased

Declaration of Competing Interest

None.

Acknowledgments

The authors thank Ethan Benevides and Song M. Kim for technical assistance. Research reported here was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103423.

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