Hypoxic Ventilatory Response In Rats Research Articles

Orexin facilitates the hypoxic ventilatory response in rats

Orexin is a hypothalamic neuropeptide involved in a variety of functions including arousal, body temperature regulation, and feeding. Orexin neurons are active in wakefulness (or the dark phase in rats) and mostly silent in sleep (light phase), implying the effects of orexin are greater in wakefulness and/or dark phase. Recent evidence implicates orexin in the control of cardiorespiratory function, including the respiratory response to CO2 and blood pressure regulation. Whether orexin influences normal breathing in a state-dependent manner is unresolved. In addition, although the role of orexin in the hypercapnic ventilatory response is well established, its effects on the acute hypoxic ventilatory response (HVR) has not been investigated. We hypothesized that orexin receptor blockade would lead to hypoventilation in wakefulness but not sleep and would reduce the HVR. To test these hypotheses, we conducted two separate experiments, both of which were performed in the light phase. First, we used whole-body plethysmography to measure the ventilation of adult rats in room air. Rats were implanted with EEG and EMG electrodes to determine vigilance state, which was confirmed by applying Fast Fourier Transform on the EEG signals. After a 1hr baseline period, we injected i.p. either suvorexant (an OxR1 and OxR2 antagonist, 20 mg/kg i.p.) or vehicle (100% DMSO, i.p.) and allowed the rats to freely behave for 4 hours in the chamber while breathing room air. Separate groups of rats, also instrumented with EEG and EMG electrodes, were exposed to graded hypoxia (15%, 13%, 11%, and 9% O2) following administration of either 20 mg/kg i.p. suvorexant or vehicle. We found that, irrespective of state of vigilance, OxR1 and OxR2 blockade had no effect on baseline breathing, but significantly reduced the HVR (treatment × O2 level: p=0.03). The effect on the HVR was especially evident at 13% and 11% O2 when the response was reduced by ~30%. We show that orexin facilitates the HVR in the light phase, even though the orexin neurons are silent. Whether orexin has a larger effect on the HVR during the dark phase, when orexin neurons are active, has yet to be determined.

Effects of A2a Receptors on the Hypoxic Ventilatory Response in Rats Exposed to Chronic Sustained Hypoxia

Recent studies show that long term facilitation of phrenic nerve activity (LTF) with acute intermittent hypoxia (IH) involves two signaling pathways. In addition to the well‐known serotonergic (Gq) pathway, a Gs pathway can be activated by adenosine receptor 2 (A2a) agonists and cause LTF without IH (J Neurosci 28:2033‐42, 2008). Gs can inhibit the Gq pathway as A2a antagonists increase LTF from IH by disinhibiting Gq (J Physiol 588:255‐66, 2010). We tested the hypothesis that Gs also contributes to plasticity in ventilatory control during chronic sustained hypoxia (CH) by measuring the effects of A2a antagonists and agonists on ventilatory responses to hypoxia and hypercapnia in adult male rats before and after one week of CH (PO2 = 80 Torr in a hypobaric chamber). Ventilation measured in awake unrestrained rats with barometric pressure plethysmography increased after CH; acute ventilatory responses to hypoxia or hypercapnia increased also. Acute administration of A2a antagonist (MSX‐3, 1 mg/Kg i.p.) after CH did not change ventilation or acute ventilatory responses. A2a agonists (CGS 21680, 100 µg/Kg i.p.) before CH had no effect on ventilation or ventilatory responses, in contrast to stimulation reported for sleeping rats (J Neurosci 28:2033‐42, 2008). The results do not support a role for Gs in the increased ventilatory drive and hypoxic responses with CH, in contrast to their role in LTF in IH.Supported by NIH RO1 HL‐081823.

Selecting representative ages for developmental changes of respiratory irregularities and hypoxic ventilatory response in rats

Apnea frequency and the weak ventilatory response to hypoxia are a major clinical correlates of the immaturity of respiratory control system in preterm neonates. Rats are frequently used as model to study the respiratory control during development. However, little is known about the postnatal ages that best represent these respiratory irregularities and the hypoxic ventilatory response. Using plethysmography, we assessed baseline minute ventilation, ventilatory response to moderate hypoxia (FiO2 = 12%, 20 min) and apnea frequency in awake and non-anesthetized rats at the postnatal ages of 1, 4, 7, 12, 21 and 90 days old (P1, P4, P7, P12, P21, and P90, respectively). Baseline minute ventilation slightly increased in P4 (~25% vs P1) then gradually decreased with age (age effect: p < 0.05). The lowest level of ventilation was observed in P90 (p < 0.01 vs all ages). Minute ventilation (% from baseline) in response to hypoxia showed the well-known biphasic pattern in all rats at 12 days old or less. Minute ventilation at the initial phase of the hypoxic response was not significantly different between P1, P4, between P7, P12 and between P21, P90. The late phase of the hypoxic response was similar between P1, P4, and between P21, P90, but was significantly different between P7 and P12 (p < 0.05). Under baseline or hypoxic condition, the higher number of apnea frequency (spontaneous and post- sigh) was observed in P1, it then decreased progressively with age (age effect: p < 0.01 for baseline; p < 0.001 for hypoxia). These results suggest that when P4, P7 and P12 are selected to represent the age-dependent changes of the hypoxic ventilatory response in rats, the P1 rats should be included to better describe the age-dependence of apnea frequency.

The acute hypoxic ventilatory response under halothane, isoflurane, and sevoflurane anaesthesia in rats*

The relative order of potency of anaesthetic agents on the hypoxic ventilatory response has been tested in humans, but animal data are sparse. We examined the effects of 1.4, 1.6, 1.8, and 2.0 MAC halothane, isoflurane, and sevoflurane on phrenic nerve activity in euoxia (baseline) and during acute normocapnic hypoxia (inspired oxygen fraction 0.09) in adult male Sprague-Dawley rats. With halothane, all animals became apnoeic even in euoxia, and the hypoxic response was completely abolished at all anaesthetic levels. With isoflurane, 5 of 14 animals exhibited phrenic nerve activity in euoxia at 1.4 MAC and demonstrated a hypoxic response (302% of baseline activity), but all became apnoeic and lost the hypoxic response at higher doses. With sevoflurane, phrenic nerve activity and a hypoxic response was preserved in at least some animals at all doses (i.e. even the highest dose of 2.0 MAC). Similar to the rank order of potency previously observed in humans, the relative order of potency of depression of the hypoxic ventilatory response in rats was halothane (most depressive) > isoflurane > sevoflurane (p = 0.01 for differences between agents).

Neonatal intermittent hypoxia induces sex‐specific enhancement of hypoxic ventilatory response in rat pups

We tested the hypothesis that chronic intermittent hypoxia induces gender‐specific effects on hypoxic ventilatory response (HVR) in newborn rats. Rat pups were exposed with their dams to normal room air (controls) or to chronic intermittent hypoxia (IH: O2 levels cycled between 21% and 5% every 10 minutes for 1h followed by 1h under normoxia, 24h/day) during postnatal days 1–10. Minute ventilation (VE) was measured in 10‐day‐old male and female rat pups using whole‐body plethysmography. To assess HVR, VE was measured in normoxia and during acute hypoxia (5% achieved in 60 seconds), and expressed as % changes vs. baseline. IH induced a growth‐delay of similar magnitude in males and females. Under normoxia, respiratory frequency was slightly lower in both male and female IH rats vs. controls, but VE was similar. In males, HVR was higher in IH rats (+57±13%) vs. controls (+32±8%, p=0,001). In females, HVR was similar between neonatal IH (+35±13%) and control rats (+42±7%, p=ns). This study shows that neonatal IH increases HVR specifically in male rat pups, and suggests that apneas may have sex‐specific effects on respiratory control in neonates. (Founded by FRSQ, Sick kids foundation/CIHR).

Perinatal hyperoxia alters the expression of neurotrophic factors and their receptors in the rat carotid body

Perinatal hyperoxia attenuates the hypoxic ventilatory response in rats by altering development of the carotid body and its afferent neurons. We hypothesized that these changes in carotid body growth and innervation involve changes in the expression of neurotrophic factors or their receptors. Rats were born and raised in hyperoxia (60% O2) or normoxia (21% O2) until studied at 1–14 days of age (i.e., P1–P14). Carotid body volume was 30% smaller in hyperoxia‐reared rats than their normoxia‐reared counterparts by P4 (P=0.02) and showed little growth thereafter. In contrast, carotid bodies of normoxia‐reared rats more than doubled in size between P4 and P14. We measured mRNA expression for brain‐derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (Trk B), and glial cell line‐derived neurotrophic factor (GDNF) and its receptor Ret in carotid bodies at P3 via quantitative RT‐PCR. Ret expression was reduced ~30% in hyperoxia‐treated rats (P&lt;0.01), but no differences were detected for BDNF, TrkB, or GDNF. In addition, BDNF and GDNF protein expression levels were measured at P3 via ELISA. BDNF was reduced by ~70% in the carotid bodies of hyperoxia‐treated rats (P=0.02). GDNF appeared to decrease, depending on normalization methods. These data suggest a role for neurotrophic factors in hyperoxia‐induced respiratory plasticity. Supported by NIH grant P20 RR‐016463 (Maine INBRE).

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.

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.

Neonatal maternal separation and early life programming of the hypoxic ventilatory response in rats

The neonatal period is critical for central nervous system (CNS) development. Recent studies have shown that this basic neurobiological principle also applies to the neural circuits regulating respiratory activity as exposure to excessive or insufficient chemosensory stimuli during early life can have long-lasting consequences on the performance of this vital system. Although the tactile, olfactory, and auditory stimuli that the mother provides to her offspring during the neonatal period are not directly relevant to respiratory homeostasis, they likely contribute to respiratory control development. This review outlines the rationale for the link between maternal stimuli and programming of the hypoxic ventilatory response during early life, and presents recent results obtained in rats indicating that experimental disruption of mother-pup interaction during this critical period elicits significant phenotypic plasticity of the hypoxic ventilatory response.

Release of ATP in the ventral medulla during hypoxia in rats: role in hypoxic ventilatory response.

P2X2 receptor subunits of the ATP-gated ion channels are expressed by physiologically identified respiratory neurons in the ventral respiratory column, implicating ATP in the control of respiratory activity. We now show that, during hypoxia, release of ATP in the ventrolateral medulla (VLM) plays an important role in the hypoxic ventilatory response in rats. By measuring ATP release in real time at the ventral surface of the medulla with novel amperometric biosensors, we found that hypoxia (10% O2; 5 min) induced a marked increase in the concentration of ATP (approximately 3 microm). This ATP release occurred after the initiation of enhanced respiratory activity but coincided with the later hypoxia-induced slowing of the respiratory rhythm. ATP was also released at the ventral surface of the medulla during hypoxia in peripherally chemodenervated animals (vagi, aortic, and carotid sinus nerve sectioned). By using horizontal slices of the rat medulla, we found that, during hypoxia, ATP is produced throughout the VLM in the locations corresponding to the ventral respiratory column. Blockade of ATP receptors in the VLM (microinjection of P2 receptor antagonist pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid; 100 mum) augmented the hypoxia-induced secondary slowing of the respiratory rhythm. Our findings suggest that ATP released within the ventral respiratory column is involved in maintenance of the respiratory activity in conditions when hypoxia-induced slowing of respiration occurs. These data illustrate a new functional role for ATP-mediated purinergic signaling in the medullary mechanisms controlling respiratory activity.

Respiratory insensitivity to acute hypoxia persisting after correction of tetralogy of Fallot.

Respiratory insensitivity to acute hypoxia persisting after correction of tetralogy of Fallot.

Irreversible respiratory insensitivity to acute hypoxia in man born at high altitude.

Irreversible respiratory insensitivity to acute hypoxia in man born at high altitude.