E. Burt Olson

Chemoafferent degeneration and carotid body hypoplasia following chronic hyperoxia in newborn rats

1 To define the role of environmental oxygen in regulating postnatal maturation of the carotid body afferent pathway, light and electron microscopic methods were used to compare chemoafferent neurone survival and carotid body development in newborn rats reared from birth in normoxia (21 % O2) or chronic hyperoxia (60 % O2). 2 Four weeks of chronic hyperoxia resulted in a significant 41 % decrease in the number of unmyelinated axons in the carotid sinus nerve, compared with age‐matched normoxic controls. In contrast, the number of myelinated axons was unaffected by hyperoxic exposure. 3 Chemoafferent neurones, located in the glossopharyngeal petrosal ganglion, already exhibited degenerative changes following 1 week of hyperoxia from birth, indicating that even a relatively short hyperoxic exposure was sufficient to derange normal chemoafferent development. In contrast, no such changes were observed in the vagal nodose ganglion, demonstrating that the effect of high oxygen levels was specific to sensory neurones in the carotid body afferent pathway. Moreover, petrosal ganglion neurones were sensitive to hyperoxic exposure only during the early postnatal period. 4 Chemoafferent degeneration in chronically hyperoxic animals was accompanied by marked hypoplasia of the carotid body. In view of previous findings from our laboratory that chemoafferent neurones require trophic support from the carotid body for survival after birth, we propose that chemoafferent degeneration following chronic hyperoxia is due specifically to the loss of target tissue in the carotid body.

Phrenic responses to isocapnic hypoxia in adult rats following perinatal hyperoxia

The purpose of this study was to test the hypothesis that carotid body-mediated, phrenic nerve responses to hypoxia are attenuated in adult rats that had been previously exposed to perinatal hyperoxia (one month of 60% O2; perinatal treated rats.) Integrated phrenic nerve responses to strictly controlled isocapnic hypoxia were measured in urethane-anesthetized, vagotomized, paralyzed and ventilated adult rats 2-5 months after perinatal hyperoxia, before and after bilateral carotid denervation. In untreated control rats, phrenic burst frequency, peak amplitude of integrated phrenic activity and minute phrenic activity increased 21 +/- 3 bursts/min (mean +/- SE), 158 +/- 20% and 279 +/- 34%, respectively, during hypoxia (50 Torr PaO2). In contrast, phrenic nerve activity increased to a significantly lesser degree in perinatal treated rats (frequency, 12 +/- 2 bursts/min; amplitude, 87 +/- 13%; minute activity, 150 +/- 19%; all P < 0.05). Hypoxic phrenic responses were abolished by carotid degeneration in both rat groups. In rats exposed to hyperoxia as adults, hypoxic phrenic responses were not attenuated versus untreated control rats. The data indicate that carotid body-mediated, isocapnic hypoxic chemoreflexes are impaired in perinatal treated rats, an effect unique to development. These effects cannot be accounted for by differences in blood gases (O2 or CO2) or pulmonary mechanics.

Xanthine oxidase inhibition attenuates endothelial dysfunction caused by chronic intermittent hypoxia in rats.

Background: Xanthine oxidase is a major source of superoxide in the vascular endothelium. Previous work in humans demonstrated improved conduit artery function following xanthine oxidase inhibition in patients with obstructive sleep apnea. Objectives: To determine whether impairments in endothelium-dependent vasodilation produced by exposure to chronic intermittent hypoxia are prevented by in vivo treatment with allopurinol, a xanthine oxidase inhibitor. Methods: Sprague-Dawley rats received allopurinol (65 mg/kg/day) or vehicle via oral gavage. Half of each group was exposed to intermittent hypoxia (FIO2 = 0.10 for 1 min, 15×/h, 12 h/day) and the other half to normoxia. After 14 days, gracilis arteries were isolated, cannulated with micropipettes, and perfused and superfused with physiological salt solution. Diameters were measured before and after exposure to acetylcholine (10–6M) and nitroprusside (10–4M). Results: In vehicle-treated rats, intermittent hypoxia impaired acetylcholine-induced vasodilation compared to normoxia (+4 ± 4 vs. +21 ± 6 µm, p = 0.01). Allopurinol attenuated this impairment (+26 ± 6 vs. +34 ± 9 µm for intermittent hypoxia and normoxia groups treated with allopurinol, p = 0.55). In contrast, nitroprusside-induced vasodilation was similar in all rats (p = 0.43). Neither allopurinol nor intermittent hypoxia affected vessel morphometry or systemic markers of oxidative stress. Urinary uric acid concentrations were reduced in allopurinol- versus vehicle-treated rats (p = 0.02). Conclusions: These data confirm previous findings that exposure to intermittent hypoxia impairs endothelium-dependent vasodilation in skeletal muscle resistance arteries and extend them by demonstrating that this impairment can be prevented with allopurinol. Thus, xanthine oxidase appears to play a key role in mediating intermittent hypoxia-induced vascular dysfunction.

Responses of single‐unit carotid body chemoreceptors in adult rats

1 Our goal was to describe the in situ responses in rats of single‐unit carotid body chemoreceptors to changes in arterial PO2 and PCO2. We identified single‐unit carotid chemoreceptor activity in male, adult Sprague‐Dawley rats by their rapid responses to i.v. NaCN (20 μg) and transient (10 s) asphyxia. 2 Single‐unit chemoreceptor responses to isocapnic changes in oxygenation within the arterial oxygen pressure range 34‐114 mmHg were described by the power function: fdis= 74010(P  a,O 2 )−2.5; (r2= 0.6), where fdis is the discharge frequency (spikes s−1), P  a,O 2 is the arterial oxygen partial pressure (mmHg) and r2 is the correlation coefficient. 3 The responses to iso‐oxic changes in CO2, assumed to be linear, had a slope of 0.089 spikes s−1 (mmHg Pa,CO2)−1 (r2= 0.7). 4 We conclude that carotid body chemoreceptors in adult rats have responses to changes in P  a,O 2 and Pa,CO2 similar to those of other species.

Ventilatory adaptation to hypoxia occurs in serotonin-depleted rats

To test the hypothesis that serotonin mediated respiratory activity is involved in ventilatory adaptation to hypoxia, rats were treated with parachlorophenylalanine (PCPA), a potent, long-acting inhibitor of tryptophan hydroxylase, the rate-limiting enzyme in the biosynthesis of serotonin. In normoxia, a single, intraperitoneal injection of 300 mg PCPA/kg body weight decreased the Paco2 from a control level at 39.1 +/- 0.6 Torr (mean +/- 95% confidence limits) to 34.0 +/- 0.6 Torr measured during a period from 1 to 48 h following PCPA treatment. This PCPA-produced hyperventilation corresponds to an increase of 3.7 +/- 0.5 in the VA (BTPS)/Vco2 (STPD) ratio. Hyperventilation during ventilatory adaptation to hypoxia (PIO2 approximately equal to 90 Torr) was superimposed in an additive fashion on the underlying hyperventilation due to PCPA pretreatment. Specifically, PCPA pretreatment caused an average 3.5 +/- 1.2 increase in the VA/VCO2 ratio determined in acute (1 h) hypoxia, chronic (24 h) hypoxia and acute return to normoxia following chronic hypoxia. Since ventilatory adaptation to hypoxia occurred in rats treated with PCPA, the prolonged, serotonin mediated respiratory activity described by Millhorn et al. (1980b) is probably not important in ventilatory acclimatization to - or deacclimatization from - hypoxia.

Time course of intermittent hypoxia-induced impairments in resistance artery structure and function

We previously demonstrated that chronic exposure to intermittent hypoxia (CIH) impairs endothelium-dependent vasodilation in rats. To determine the time course of this response, rats were exposed to CIH for 3, 14, 28, or 56 days. Then, we measured acetylcholine- and nitroprusside-induced vasodilation in isolated gracilis arteries. Also, we measured endothelial and inducible nitric oxide synthase, nitrotyrosine, and collagen in the arterial wall and urinary isoprostanes. Endothelium-dependent vasodilation was impaired after 2 weeks of CIH. Three days of CIH was not sufficient to produce this impairment and longer exposures (i.e. 4 and 8 weeks) did not exacerbate it. Impaired vasodilation was accompanied by increased collagen deposition. CIH elevated urinary isoprostane excretion, whereas there was no consistent effect on either isoform of nitric oxide synthase or nitrotyrosine. Exposure to CIH produces functional and structural deficits in skeletal muscle resistance arteries. These impairments develop within 2 weeks after initiation of exposure and they are accompanied by systemic evidence of oxidant stress.

Time-dependent adaptation in the hemodynamic response to hypoxia

In rats, acute exposure to hypoxia causes a decrease in mean arterial pressure (MAP) caused by a predominance of hypoxic vasodilation over chemoreflex-induced vasoconstriction. We previously demonstrated that exposure to chronic intermittent hypoxia (CIH) impairs hypoxic vasodilation in isolated resistance arteries; therefore, we hypothesized that the acute systemic hemodynamic responses to hypoxia would be altered by exposure to CIH. To test this hypothesis, rats were exposed to CIH for 14 days. Heart rate (HR) and MAP were monitored by telemetry. On the first day of CIH exposure, acute episodes of hypoxia caused a decrease in MAP (-9+/-5 mmHg) and an increase in HR (+45+/-4 beats/min). On the 14th day of CIH exposure the depressor response was attenuated (-4+/-1mmHg; 44% of the day 1 response) and the tachycardia was enhanced (+68+/-2 beats/min; 151% of the day 1 response). The observed time-dependent modulation of the acute hemodynamic responses to hypoxia may reflect important changes in neurocirculatory regulation that contribute to CIH-induced hypertension.

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.

Naloxone accelerates the rate of ventilatory acclimatization to hypoxia in awake rats

During ventilatory acclimatization to hypoxia in rats, PaCO2 progressively falls from about 40 torr in normoxia (PIO2 approximately equal to 150 torr) to a new steady-state at about 23 torr in chronic hypoxia (24 or more hours at PIO2 approximately equal to 90 torr). In acute (20 or 60 minutes) hypoxia naloxone treatment caused a hyperventilation greater than that caused by acute hypoxia alone. Following 20 minutes hypoxia, naloxone treated rats had a PaCO2 = 28.6 +/- 0.7 torr (mean +/- 95% confidence limits) which was significantly lower (P less than .001) than the saline treated PaCO2 = 31.0 +/- 0.6 torr. In contrast, in normoxia and at 24 hour hypoxia and at 20 minute return to normoxia following 24 hours hypoxia, naloxone treatment had no effect on PaCO2. We conclude that in the rat about one third of the ventilatory acclimatization to hypoxia is due to a progressively decreasing endogenous opioid-like inhibition of ventilation.

Expression of 5-HT3 receptors in primary sensory neurons of the petrosal ganglion of adult rats.

By using a specific antiserum, expression of the 5-HT3 receptor was examined in the petrosal ganglion (PG) of adult male rats. We found that the 5-HT3 receptors are widely distributed in the PG. This finding was confirmed by RT-PCR detection of the 5-HT3 receptor mRNA in the tissue. Unlike the distribution patterns of tyrosine hydroxylase (TH), which occurred in limited regions of PG, the 5-HT3 receptors seemed to distribute throughout the ganglion. As many TH-positive neurons in PG innervate type I cells in the carotid body, the coexistence of 5-HT3 receptor and TH in some neurons suggests that this receptor may play a role in carotid body chemoreception.