Carotid sinus nerve responses and ventilatory acclimatization to hypoxia in adult rats following 2 weeks of postnatal hyperoxia

Adult rats have decreased carotid body volume and reduced carotid sinus nerve, phrenic nerve, and ventilatory responses to acute hypoxic stimulation after exposure to postnatal hyperoxia (60% O2, PNH) during the first 4 weeks of life. Moreover, sustained hypoxic exposure (12%, 7 days) partially reverses functional impairment of the acute hypoxic phrenic nerve response in these rats. Similarly, 2 weeks of PNH results in the same phenomena as above except that ventilatory responses to acute hypoxia have not been measured in awake rats. Thus, we hypothesized that 2-week PNH-treated rats would also exhibit blunted chemoafferent responses to acute hypoxia, but would exhibit ventilatory acclimatization to sustained hypoxia. Rats were born into, and exposed to PNH for 2 weeks, followed by chronic room-air exposure. At 3-4 months of age, two studies were performed to assess: (1) carotid sinus nerve responses to asphyxia and sodium cyanide in anesthetized rats and (2) ventilatory and blood gas responses in awake rats before (d0), during (d1 and d7), and 1 day following (d8) sustained hypoxia. Carotid sinus nerve responses to i.v. NaCN and asphyxia (10 s) were significantly reduced in PNH-treated versus control rats; however, neither the acute hypoxic ventilatory response nor the time course or magnitude of ventilatory acclimatization differed between PNH and control rats despite similar levels of PaO2 . Although carotid body volume was reduced in PNH rats, carotid body volumes increased during sustained hypoxia in both PNH and control rats. We conclude that normal acute and chronic ventilatory responses are related to retained (though impaired) carotid body chemoafferent function combined with central neural mechanisms which may include brainstem hypoxia-sensitive neurons and/or brainstem integrative plasticity relating both central and peripheral inputs.     

The influence of chronic hypoxia upon chemoreception

Carotid body chemoreceptors are essential for time-dependent changes in ventilatory control during chronic hypoxia. Early theories of ventilatory acclimatization to hypoxia focused on time-dependent changes in known ventilatory stimuli, such as small changes in arterial pH that may play a significant role in some species. However, plasticity in the cellular and molecular mechanisms of carotid body chemoreception play a major role in ventilatory acclimatization to hypoxia in all species studied. Chronic hypoxia causes changes in (a) ion channels (potassium, sodium, calcium) to increase glomus cell excitability, and (b) neurotransmitters (dopamine, acetylcholine, ATP) and neuromodulators (endothelin-1) to increase carotid body afferent activity for a given PO(2) and optimize O(2)-sensitivity. O(2)-sensing heme-containing molecules in the carotid body have not been studied in chronic hypoxia. Plasticity in medullary respiratory centers processing carotid body afferent input also contributes to ventilatory acclimatization to hypoxia. It is not known if the same mechanisms occur in patients with chronic hypoxemia from lung disease or high altitude natives.     

Developmental plasticity of the hypoxic ventilatory response in rats induced by neonatal hypoxia

Neonatal hypoxia alters the development of the hypoxic ventilatory response in rats and other mammals. Here we demonstrate that neonatal hypoxia impairs the hypoxic ventilatory response in adult male, but not adult female, rats. Rats were raised in 10% O₂ for the first postnatal week, beginning within 12 h after birth. Subsequently, ventilatory responses were assessed in 7- to 9-week-old unanaesthetized rats via whole-body plethysmography. In response to 12% O₂, male rats exposed to neonatal hypoxia increased ventilation less than untreated control rats (mean ± s.e.m. 35.2 ± 7.7% versus 67.4 ± 9.1%, respectively; P = 0.01). In contrast, neonatal hypoxia had no lasting effect on hypoxic ventilatory responses in female rats (67.9 ± 12.6% versus 61.2 ± 11.7% increase in hypoxia-treated and control rats, respectively; P > 0.05). Normoxic ventilation was unaffected by neonatal hypoxia in either sex at 7–9 weeks of age ( P > 0.05). Since we hypothesized that neonatal hypoxia alters the hypoxic ventilatory response at the level of peripheral chemoreceptors or the central neural integration of chemoafferent activity, integrated phrenic responses to isocapnic hypoxia were investigated in urethane-anaesthetized, paralysed and ventilated rats. Phrenic responses were unaffected by neonatal hypoxia in rats of either sex ( P > 0.05), suggesting that neonatal hypoxia-induced plasticity occurs between the phrenic nerve and the generation of airflow (e.g. neuromuscular junction, respiratory muscles or respiratory mechanics) and is not due to persistent changes in hypoxic chemosensitivity or central neural integration. The basis of sex differences in this developmental plasticity is unknown.     

Recovery of carotid body O2 sensitivity following chronic postnatal hyperoxia in rats

Chronic postnatal hyperoxia blunts the hypoxic ventilatory response (HVR) in rats, an effect that persists for months after return to normoxia. To determine whether decreased carotid body O(2) sensitivity contributes to this lasting impairment, single-unit chemoafferent nerve and glomus cell calcium responses to hypoxia were recorded from rats reared in 60% O(2) through 7d of age (P7) and then returned to normoxia. Single-unit nerve responses were attenuated by P4 and remained low through P7. After return to normoxia, hypoxic responses were partially recovered within 3d and fully recovered within 7-8d (i.e., at P14-15). Glomus cell calcium responses recovered with a similar time course. Hyperoxia altered carotid body mRNA expression for O(2)-sensitive K(+) channels TASK-1, TASK-3, and BK(Ca), but only TASK-1 mRNA paralleled changes in chemosensitivity (i.e., downregulation by P7, partial recovery by P14). Collectively, these data do not support a role for reduced O(2) sensitivity of individual chemoreceptor cells in long-lasting reduction of the HVR after developmental hyperoxia.     

Ventilation- and carotid chemoreceptor discharge-response to hypoxia during induced hypothermia in halothane anesthetized rat

It has been hypothesized that respiratory "gain" to hypoxic stimulus is not depressed in hypothermic animals though ventilation and that metabolic O(2) demand (Vo(2)) decreases with reduction in body temperature. The present study addressed this hypothesis by quantitative analysis of ventilatory and carotid chemoreceptor responsiveness to hypoxia during induced hypothermia in halothane anesthetized and spontaneously breathing rats. Rectal temperature was lowered from 37 degrees C (normothermia) to 30 and 25 degrees C by cooling body surface at comparable anesthetic depth without inducing shivering. Ventilation (V(E)), V(O2), PaO(2) and carotid chemoreceptor afferent discharges were measured during hyperoxic and hypoxic gas breathing. PaO(2) values at the same Fi(O2) (range 0. 35-0.08) decreased progressively as rectal temperature decreased. Both the V(E)/V(O2)- and chemoreceptor discharge-response curves shifted toward a lower PaO(2) range with a slight increase in the response slopes during hypothermia. The results indicated that the sensitivity of carotid chemoreceptor and ventilatory responses to hypoxia did not decrease at reduced body temperature. It is concluded that carotid chemoreceptor mediated regulation of ventilation is tightly coupled to changes in PaO(2 )range in halothane anesthetized rats during induced hypothermia.     

Intermittent hypoxia and plasticity of respiratory chemoreflexes in metamorphic bullfrog tadpoles

We tested the hypothesis that intermittent hypoxia elicits plasticity in respiratory chemoreflexes in bullfrog tadpoles. Metamorphic tadpoles (Taylor-Kollros stages XVI-XX) were subjected to intermittent hypoxia (PW(O(2))=45 Torr; 12 h/day) or constant normoxia (PW(O(2))=156 Torr) for 2 weeks before ventilatory responses to hypoxia and hypercarbia were measured. Buccal pressure changes were used to quantify the frequency and amplitude of movements associated with gill and lung ventilation. Morphometric assessment showed that intermittent hypoxia delayed development in comparison with controls. Oxygen consumption was enhanced in tadpoles subjected to intermittent hypoxia; however, this increase was not sufficient to affect basal ventilatory activity or the hypoxic ventilatory response. During acute hypercarbic exposure, tadpoles subjected to intermittent hypoxia showed (1) a greater decrease in gill ventilation frequency and (2) a greater increase in lung ventilation frequency than tadpoles maintained under control conditions. We conclude that intermittent hypoxia augments the responsiveness to hypercarbia, thereby promoting lung ventilation when animals face this stimulus. This manifestation of respiratory plasticity may reflect uncoupling between physiological and morphological development in the bi-modally breathing bullfrog tadpole.     

Respiratory, cerebrovascular and cardiovascular responses to isocapnic hypoxia

We simultaneously measured respiratory, cerebrovascular and cardiovascular responses to 10-min of isoxic hypoxia at three constant CO(2) tensions in 15 subjects. We observed four response patterns, some novel, for ventilation, middle cerebral artery blood flow velocity, heart rate and mean arterial blood pressure. The occurrence of the response patterns was correlated between some measures. Isoxic hyperoxic and hypoxic ventilatory sensitivities to CO(2) derived from these responses were equivalent to those measured with modified (Duffin) rebreathing tests, but cerebrovascular sensitivities were not. We suggest the different ventilatory response patterns reflect the time course of carotid body afferent activity; in some individuals, carotid body function changes during hypoxia in more complex ways than previously thought. We concluded that isoxic hyperoxic and hypoxic ventilatory sensitivities to CO(2) can be measured using multiple hypoxic ventilatory response tests only if care is taken choosing the isocapnic CO(2) levels used, but a similar approach to measuring the cerebrovascular response to isocapnic hyperoxia and hypoxia is unfeasible.     

Dopaminergic mechanisms of neural plasticity in respiratory control: transgenic approaches

Data supporting the hypothesis that dopamine-2 receptors (D(2)-R) contribute to time-dependent changes in the hypoxic ventilatory response (HVR) during acclimatization to hypoxia are briefly reviewed. Previous experiments with transgenic animals (D(2)-R 'knockout' mice) support this hypothesis (J. Appl. Physiol. 89 (2000) 1142). However, those experiments could not determine (1) if D(2)-R in the carotid body, the CNS, or both were involved, or (2) if D(2)-R were necessary during the acclimatization to hypoxia versus some time prior to chronic hypoxia, e.g. during a critical period of development. Additional experiments on C57BL/6J mice support the idea that D(2)-R are critical during the period of exposure to hypoxia for normal ventilatory acclimatization. D(2)-R in carotid body chemoreceptors predominate under control conditions to inhibit normoxic ventilation, but excitatory effects of D(2)-R, presumably in the CNS, predominate after acclimatization to hypoxia. The inhibitory effects of D(2)-R in the carotid body are reset to operate primarily under hypoxic conditions in acclimatized rats, thereby optimizing O(2)-sensitivity.     

Enhancement of the breathing frequency response to hypoxia by neonatal caffeine treatment in adult male rats: The role of testosterone

Caffeine is a common treatment for apnea of prematurity. Although relatively safe, little is known about the potential long-term effects of this treatment on respiratory control development. We previously showed that adult male (but not female) rats previously subjected to neonatal caffeine treatment (NCT; 15 mg/kg/day, postnatal days 3–12) show a higher breathing frequency response during the early phase of hypoxic exposure. To address the role of sexual hormones in this sexual dimorphism, the present study tested the hypothesis that in adult male rats, circulating testosterone contributes to NCT-related augmentation of the acute breathing frequency response to hypoxia. Whole body plethysmography was used to compare the acute ventilatory response to moderate hypoxia (FIO₂ = 0.12; 20 min) between rats previously subjected to NCT or neonatal water treatment (NWT; same treatment as NCT but using water). In each group, rats were either sham-operated or gonadectomized (GDX) 14 days prior to ventilatory measurements. In sham-operated rats, the increase in breathing frequency measured during the first 8 min of hypoxia was greater in NCT rats versus NWT. The hypoxic ventilatory response measured at the end of the hypoxia was not affected by treatment, thus indicating that NCT mainly affected the peripheral component of the chemoreflex. Gonadectomy had no effect on NCT but augmented the frequency response of NWT rats to the same level of NCT, thus eliminating the between-group difference. NCT may interfere with the inhibitory effect of circulating testosterone on carotid body function. Although appealing, additional experiments are necessary to substantiate this interpretation.     

Respiratory plasticity after perinatal hyperoxia is not prevented by antioxidant supplementation

Perinatal hyperoxia attenuates the hypoxic ventilatory response in rats by altering development of the carotid body and its chemoafferent neurons. In this study, we tested the hypothesis that hyperoxia elicits this plasticity through the increased production of reactive oxygen species (ROS). Rats were born and raised in 60% O(2) for the first two postnatal weeks while treated with one of two antioxidants: vitamin E (via milk from mothers whose diet was enriched with 1000 IU vitamin E kg(-1)) or a superoxide dismutase mimetic, manganese(III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP; via daily intraperitoneal injection of 5-10 mg kg(-1)); rats were subsequently raised in room air until studied as adults. Peripheral chemoreflexes, assessed by carotid sinus nerve responses to cyanide, asphyxia, anoxia and isocapnic hypoxia (vitamin E experiments) or by hypoxic ventilatory responses (MnTMPyP experiments), were reduced after perinatal hyperoxia compared to those of normoxia-reared controls (all P<0.01); antioxidant treatment had no effect on these responses. Similarly, the carotid bodies of hyperoxia-reared rats were only one-third the volume of carotid bodies from normoxia-reared controls (P <0.001), regardless of antioxidant treatment. Protein carbonyl concentrations in the blood plasma, measured as an indicator of oxidative stress, were not increased in neonatal rats (2 and 8 days of age) exposed to 60% O(2) from birth. Collectively, these data do not support the hypothesis that perinatal hyperoxia impairs peripheral chemoreceptor development through ROS-mediated oxygen toxicity.     

Developmental hyperoxia attenuates the hypoxic ventilatory response in Japanese quail (Coturnix japonica)

Early life experiences can influence development of the respiratory control system. We hypothesized that chronic hyperoxia (60% O(2)) during development would attenuate the hypoxic ventilatory response (HVR) of Japanese quail (Coturnix japonica), similar to the effects of developmental hyperoxia in mammals. Quail were exposed to hyperoxia during prenatal development, during postnatal development, or during both prenatal and postnatal development (for approximately 2 or 4 weeks). HVR (11% O(2)) was subsequently assessed in adults (>6 weeks old) via barometric plethysmography and compared to quail raised in normoxia (i.e., control). The HVR of quail exposed to hyperoxia both prenatally and postnatally was reduced 50-60% compared to control quail whereas postnatally exposed quail exhibited normal HVR. The effects of prenatal hyperoxia on HVR were equivocal and depended on how HVR was expressed. We conclude that developmental exposure to 60% O(2) attenuates the HVR in quail and that the critical period for this plasticity encompasses the late prenatal and early postnatal periods.     

Postnatal development of carotid body glomus cell O2 sensitivity

In mammals, the main sensors of arterial oxygen level are the carotid chemoreceptors, which exhibit low sensitivity to hypoxia at birth and become more sensitive over the first few days or weeks of life. This postnatal increase in hypoxia sensitivity of the arterial chemoreceptors, termed "resetting", remains poorly understood. In the carotid body, hypoxia is transduced by glomus cells, which are secretory sensory neurons that respond to hypoxia at higher P(O2) levels than non-chemoreceptor cell types. Maturation or resetting of carotid body O2 sensitivity potentially involves numerous aspects of the O2 transduction cascade at the glomus cell level, including glomus cell neurotransmitter secretion, neuromodulator function, neurotransmitter receptor expression, glomus cell depolarization in response to hypoxia, [Ca2+]i responses to hypoxia, K+ and Ca2+ channel O2 sensitivity and K+ channel expression. However, although progress has been made in the understanding of carotid body development, the precise mechanisms underlying postnatal maturation of these numerous aspects of chemotransduction remain obscure.     

Influence of temperature on metabolism and breathing during mammalian ontogenesis

A literature survey of the ventilatory responses to changes in ambient temperature (T) in neonatal mammals reveals that, as in adults, the metabolic response to T is the major determining factor. In fact, the newborn's metabolic response to changes in T determines not only the pulmonary ventilation and the breathing pattern, but also the magnitude of the ventilatory responses to chemical stimuli and the intensity of the pulmonary reflexes at different T. The important difference from the adult is that in many neonatal mammals the control of body temperature (T(b)) is poorly developed. Hence, the metabolic response can be more similar to that of an ectothermic, rather than endothermic, animal, and T(b) can vary substantially with T. When hypoxia occurs in cold, T(b) can decrease greatly, because of the hypoxic drop in the thermoregulatory set-point, and this lowers pulmonary ventilation. Hence, in addition to the metabolic response, also the changes in T(b) are a factor modulating the ventilatory responses to T. Artificial warming of the newborn during hypoxia causes heat-dissipation responses that can be counterproductive. During ontogenesis, with prolonged cold conditions, the sustained alterations in metabolic rate and body growth do not modify the postnatal development of the respiratory control mechanisms. Presumably, this indicates that respiratory regulation develops independently from the individual's metabolic history.     

Pronounced depression by propofol on carotid body response to CO2 and K+-induced carotid body activation

Propofol is a commonly used anesthetic agent, and it attenuates hypoxic ventilatory response in humans. Propofol reduce in vivo and in vitro carotid body responses to hypoxia as well as to nicotine in experimental animals. In the present study we examined the effects of propofol on carotid body responses to hypercapnia and K(+)-induced carotid body activation and compared these effects with hypoxia in an in vitro rabbit carotid body preparation. Hypoxia, hypercapnia and potassium increased the carotid sinus nerve activity and propofol attenuated the chemoreceptor responses to all three stimuli. However, the magnitude of propofol-induced attenuation was greater for hypercapnic and K(+)-induced carotid body activation compared to the hypoxic response. These observations suggest that propofol-induced attenuation of the hypoxic response is partly secondary to depression of chemoreceptor response to hypercapnia inhibiting the synergistic interactions between O(2) and CO(2) and may involve CO(2)/H(+) sensitive K(+) channels.     

Hypoxic ventilatory responses in rats after hypercapnic hyperoxia and intermittent hyperoxia

Perinatal hyperoxia attenuates the adult hypoxic ventilatory response in rats. Hyperoxia might elicit this plasticity by inhibiting chemoreceptor activity during early life. Thus, we hypothesized that stimulating chemoreceptors with CO(2) during hyperoxia or interrupting hyperoxia with periods of normoxia would reduce the effects of hyperoxia on the hypoxic ventilatory response. Rats were born and raised in 60% O(2) for the first two postnatal weeks. Two groups were simultaneously exposed to either sustained hypercapnia (5% CO(2)) or intermittent hypercapnia (alternating 1-h exposures to 0 and 7.5% CO(2)) while another group was exposed to only intermittent hyperoxia (alternating 1-h exposures to 21 and 60% O(2)). Hypoxic ventilatory responses were assessed at 6-10 weeks of age by whole-body plethysmography. Rats exposed to intermittent hypercapnia during hyperoxia or to intermittent hyperoxia exhibited greater increases in ventilation-to-metabolism ratio ( VE/VO2 ) in response to 12.5% O(2) than rats exposed to hyperoxia alone (both P<0.05), although responses were generally less than those of normoxia-reared controls; a similar trend was observed for rats exposed to sustained hypercapnia during hyperoxia (P=0.053). These data suggest that activity-dependent mechanisms contribute to hyperoxia-induced developmental plasticity, although contributions from additional mechanisms cannot be excluded.     

Ibuprofen blocks time-dependent increases in hypoxic ventilation in rats

Recently, inflammatory processes have been shown to increase O(2)-sensitivity of the carotid body during chronic sustained hypoxia [Liu, X., He, L., Stensaas, L., Dinger, B., Fidone, S., 2009. Adaptation to chronic hypoxia involves immune cell invasion and increased expression of inflammatory cytokines in rat carotid body. Am. J. Physiol. Lung Cell Mol. Physiol. 296, L158-L166]. We hypothesized that blocking inflammation with ibuprofen would reduce ventilatory acclimatization to hypoxia by blocking such increases in carotid body O(2) sensitivity. We tested this in conscious rats treated with ibuprofen (4mg/kg IP daily) or saline during acclimatization to hypoxia ( [Formula: see text] for 7 days). Ibuprofen blocked the increase in hypoxic ventilation observed in chronically hypoxic rats treated with saline; ibuprofen had no effects on ventilation in normoxic control rats. Ibuprofen blocked increases in inflammatory cytokines (IL-1β, IL-6) in the brainstem with chronic hypoxia. The data supports our hypothesis and further analysis indicates that ibuprofen also blocks inflammatory processes in the central nervous system contributing to ventilatory acclimatization to hypoxia. Possible mechanisms linking inflammatory and hypoxic signaling are reviewed.     

Tachykinins in the control of breathing by hypoxia: pre- and post-genomic era

This article highlights major findings from physiological and pharmacological studies conducted in the pre- and post-genomic era examining the roles of substance P (SP) and other tachykinins in the response of the carotid body to hypoxia, in the ventilatory response to hypoxia and in respiratory rhythm generation. In the post-genomic period, the hypoxic ventilatory responses of mice carrying targeted deletion of genes that affect synthesis or degradation or receptor interaction of SP have been examined by us and also by other investigators. A brief summary of the findings from these investigations will also be presented. The combined observations from the pre- and post-genomic era strongly support the involvement of SP and also other tachykinins in the control of respiration during hypoxia.     

Metabolic and ventilatory sensitivity to hypoxia in avian embryos

The article discusses the establishment of pulmonary ventilation (V˙E) in the avian embryo, the metabolic and V˙E sensitivity to hypoxia and the effects of sustained embryonic hypoxia on the hatchling's V˙E chemosensitivity. Throughout embryogenesis, hypometabolism is the common response to hypoxia, with no compensation by anaerobic energy supply. It originates primarily from the depression in body growth and, later in development, from the depression of thermogenesis. The V˙E responses to acute hypoxia or hypercapnia are clearly detectable during the internal pipping phase; their magnitude rapidly increases in the first postnatal day. Sustained prenatal hypoxia diminishes the V˙E chemosensitivity of the hatchling and reduces the hypometabolic response to an acute hypoxic episode. The former most likely originates from a disturbance in the normal development of the carotid bodies, the latter from the central action of hypoxia on thermogenesis. The avian embryo is a model suitable for the studies of the development of respiratory control and offers an alternative to mammalian models for investigations on the short- and long-term effects of prenatal hypoxia.     

Erythropoietin regulates hypoxic ventilation in mice by interacting with brainstem and carotid bodies

Apart from its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions in brain, retina and heart upon ischaemic injury. However, the physiological non-erythroid functions of Epo remain unclear. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate Epo's impact on ventilation upon hypoxic exposure. Tg21 mice showed improved ventilatory response to severe acute hypoxia and moreover improved ventilatory acclimatization to chronic hypoxic exposure. Furthermore, following bilateral transection of carotid sinus nerves that uncouples the brain from the carotid body, Tg21 mice adapted their ventilation to acute severe hypoxia while chemodenervated wild-type (WT) animals developed a life-threatening apnoea. These results imply that Epo in brain modulates ventilation. Additional analysis revealed that the Epo receptor (EpoR) is expressed in the main brainstem respiratory centres and suggested that Epo stimulates breathing control by alteration of catecholaminergic metabolism in brainstem. The modulation of hypoxic pattern of ventilation after i.v. injection of recombinant human Epo in WT mice and the dense EpoR immunosignal observed in carotid bodies showed that these chemoreceptors are sensitive to plasma levels of Epo. In summary, our results suggest that Epo controls ventilation at the central (brainstem) and peripheral (carotid body) levels. These novel findings are relevant to understanding better respiratory disorders including those occurring at high altitude.