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1 Kosair Children's Hospital Research Institute, Department of Pediatrics, and 2 Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40202
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Gasping is a critically important mechanism for autoresuscitation and survival during extreme tissue hypoxia. Evidence of antecedent hypoxia in sudden infant death syndrome suggests that intermittently occurring hypoxic episodes may modify gasping and autoresuscitation. To examine this issue, an intermittent hypoxia (IH) profile consisting of alternating room air and 10% O2-balance N2 every 90 s was applied to pregnant Sprague-Dawley rats (IHRA; n = 50) and to pups after a normal pregnancy (RAIH; n = 50) as well as to control pups (RARA; n = 50). At postnatal day 5, pups were exposed to 95% N2-5% CO2, and gasping and the ability to autoresuscitate were assessed. Compared with RARA, IHRA- and RAIH-exposed pups had a reduced number of gasps, decreased overall gasp duration, and were less likely to autoresuscitate on introduction of room air to the breathing mixture during the last phase of gasping (P < 0.001 vs. RARA). We conclude that both prenatal and early postnatal IH adversely affect gasping and related survival mechanisms.
anoxic tolerance; respiration; sudden infant death syndrome; apnea
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IN THE LAST DECADE, THE INCIDENCE of sudden infant death syndrome (SIDS) has decreased primarily as a result of public campaigns aiming to reduce prone sleeping position in babies (27). However, SIDS still remains the major cause of death in apparently healthy infants. Although the precise mechanisms underlying SIDS remain undefined, one of the leading hypotheses posits that recurrent hypoxemia may precede the fatal event and affect gasping and autoresuscitation mechanisms (15, 16, 22).
When exposed to asphyxic conditions, most mammalian species will develop a transient hyperpnea, then apnea, and, within a variable period of time (gasping latency), gasping respiratory activity will emerge and continue until terminal apnea and death (2). Our laboratory has previously shown that, in developing Sprague-Dawley rats, a triphasic gasping response occurs in response to anoxia and that such response becomes monophasic at ~20 days postnatally (14). Using a similar approach, Fewell et al. (4, 5) showed that prenatal exposure to nicotine modifies anoxia-induced gasping in an age-dependent fashion and that autoresuscitation is also adversely affected. However, whether intermittent fetal or postnatal hypoxia modifies gasping responses remains unclear. We therefore hypothesized that intermittent hypoxia modifies gasping activity in the young postnatal rat and has detrimental effects on its ability to autoresuscitate.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Time-pregnant Sprague-Dawley rats were purchased from Charles River and used for all experiments. The experimental protocols were approved by the Institutional Animal Use and Care Committee and are in close agreement with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. For prenatal intermittent hypoxia exposures, pregnant rats were placed from day 5 of pregnancy until delivery in alternating room air and 10% O2-balance N2 every 90 s (IHRA; n = 6 litters). For postnatal exposures, litters were exposed within 12 h from delivery to the intermittent hypoxia profile (RAIH; n = 6 litters). As control, both pregnancy and postnatal development occurred in normoxic conditions (RARA; n = 6 litters).
Intermittent hypoxia protocol. Animals were placed in four identical commercially designed chambers (30 × 20 × 20 in.; Oxycycler model A44XO, Reming Bioinstruments, Redfield, NY), which were operated under a 12:12-h light-dark cycle (6:00 AM to 6:00 PM). Gas was circulated around each of the chambers, attached tubing, and other units at 60 l/min (i.e., 1 complete change/10 s). The O2 concentration was continuously measured by an O2 analyzer and was changed by a computerized system controlling the gas valve outlets, such that the moment-to-moment desired O2 concentration of the chamber was programmed and adjusted automatically. Deviations from the desired concentration were met by addition of N2 or O2 through solenoid valves. Ambient CO2 in the chamber was periodically monitored and maintained at <0.01% by adjusting overall chamber basal ventilation. The gas was also circulated through a molecular sieve (type 3A, Fisons, Cheshire, UK) so as to remove ammonia. Humidity was measured and maintained at 40-50% by circulating the gas through a freezer and silica gel. Ambient temperature was kept at 22-24°C. Control animals were exposed to circulating normoxic gas in one of the chambers.
Respiratory measurements. Respiratory signals elicited by displacement of the chest were acquired in the restrained animals placed in a size-appropriate head-out plethysmographic chamber (Buxco Electronics, Troy, NJ) by using the barometric method (1, 21), such that gas mixtures could be rapidly changed as needed. For all experiments, environmental temperature was maintained constant, slightly below the thermoneutral range for rat pups (30°C). Pressure changes in the chamber due to the inspiratory and expiratory displacements were measured by using a high-gain differential pressure transducer (model MP45-1, Validyne), displayed on the computer screen, and continuously digitized and stored onto a MacIntosh Personal Computer System at 125-Hz sampling frequency by using MacLab Digital Acquisition Software (ADInstruments, Castle Hill, Australia) for subsequent off-line analysis.
Experimental protocol. After a short period of acclimatization to the chamber (~15-20 min), 5-day-old pups from either experimental group were exposed to asphyxic gas (95% N2-5% CO2), and gasping was allowed to continue until death. The temporal delays in achieving a complete gas switch were ~10 s. In a subset of animals, room air was rapidly introduced during the second minute of the characteristic third phase of gasping as previously described (11, 13, 14). This phase is easily recognizable from the increased frequency of gasps (>2 gasps/min) and the exclusive presence of inspiratory efforts (6, 13, 14). The ability to autoresuscitate was documented (6, 10, 11, 13, 14, 17, 18, 23, 24).
Analysis.
Data are shown as means ± SD. The gasping variables quantitated
included gasp latency, i.e., the period of time from onset of primary
apnea and animal immobility to the first gasp, the duration of gasps,
and the gasping frequency as previously described (14).
Differences between the various treatment groups were compared by
two-way analysis of variance and the Newman-Keuls multiple-range test
for multiple comparisons. For comparisons of autoresuscitation rates,
one-way analyses of variance and 
2 analyses were conducted followed
by Fisher exact test as appropriate. A P value of <0.05 was
considered to achieve statistical significance.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IHRA pups were smaller at birth (5.4 ± 0.4 g) compared with RARA (7.2 ± 0.3 g; P < 0.001) and RAIH pups (7.0 ± 0.3 g; P < 0.001 vs. IHRA; P = not significant vs. RARA). At postnatal day 5, IHRA pups were still smaller (8.6 ± 0.5 g), and RAIH pups showed significant decreases in weight gain (7.7 ± 0.5 g) compared with RARA pups (10.7 ± 0.5 g; P < 0.001 vs. IHRA and RAIH). However, brain weights were similar for all three groups at all postnatal ages (P = not significant).
The triphasic gasping pattern occurred in all experimental groups (Fig.
1). Mean gasp latencies were similar in
RAIH (32 ± 6 s), IHRA (34 ± 8 s), and RARA pups
(30 ± 7 s; n = 20/group; P = not significant). However, the mean total gasp duration was significantly reduced in both IHRA and RAIH compared with RARA pups
(18.6 ± 3.1, 15.6 ± 2.9, and 24.7 ± 4.6 min,
respectively; Fig. 2; P < 0.001). Similarly, the overall number of gasps was diminished in
IHRA and RAIH pups (Fig. 2; P < 0.001). Indeed, the
mean total number of gasps was 31.5 ± 5.2 in IHRA, 26.2 ± 5.1 in RAIH, and 44.2 ± 7.3 in RARA pups. The mean gasp frequency was not modified during each of the gasping phases (Fig. 2).
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The ability to autoresuscitate from gasping during phase 3 was assessed
in thirty 5-day-old pups from the various litters corresponding to each
experimental group. Significantly fewer IHRA (21 of 30) and RAIH (19 of
30) pups successfully autoresuscitated compared with RARA pups (28 of
30; Fig. 3; P < 0.03 RARA vs. IHRA and P < 0.01 RARA vs. RAIH
respectively).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we show that intermittent hypoxia occurring either prenatally or during the initial days of life adversely affects gasp generating mechanisms and that the ability to autoresuscitate during the late phase of gasping is markedly curtailed.
The neural substrate for gasp generation has been assigned to a discrete region within the caudal brain stem, the lateral tegmental field of the medulla (8, 9, 25, 26). This concept has been further expanded to incorporate the possibility that the critical regions for respiratory rhythmicity may dynamically reconfigurate to generate gasping (19, 25, 26). Studies from our laboratory have previously shown that N-methyl-D-aspartate glutamate receptors are critically involved in particular components of gasp maturation (11), specifically in the early phases of respiratory activity that follow primary apnea. In addition, our laboratory has shown that increased neuronal nitric oxide synthase expression and activity occur with increasing postnatal age within the neural sites responsible for gasp generation and underlie some of the characteristic developmental changes in gasp activity (12, 13). The overall changes in N-methyl-D-aspartate receptor and neuronal nitric oxide synthase expression associated with intermittent hypoxia during fetal and early postnatal life are currently unknown.
The overall reduction in gasping duration and total gasp number as well
as the increased failure rates during autoresuscitation attempts could
reflect deficient glycogen stores. IHRA pups were smaller at birth,
and, although they demonstrated some catch-up growth after birth, they
remained smaller than controls. Similarly, RAIH pups showed lesser
weight increments than RARA, suggesting that fuel tissue reserves may
be compromised in both experimental groups. Relative depletion of
cardiac glycogen has been proposed as a potential mechanism underlying
significant differences in the autoresuscitation of SWR compared
with BALB/c weanling mice (3). Similarly, depletion of
cardiac glycogen with recurrent autoresuscitations was advanced as one
of the mechanisms potentially leading to autoresuscitation failure in
the usually successful autoresuscitating BALB/c mice (3).
However, external fuel delivery as brought about by glucose
supplementation before the asphyxic exposure exerts divergent effects
on gasping and autoresuscitation (28). Indeed, Yuan and
colleagues (28) have shown that hyperglycemia will prolong
the overall duration of gasping but reduce the frequency of
autoresuscitation. Yuan et al. (29) also examined the role of adrenergic receptors, and found that adrenalectomy shortened gasping
duration in both 1-day-old and 8-day-old rats, whereas the nonselective
-receptor antagonist phentolamine reduced the duration of gasping in
1-day-old rats but prolonged this duration in 8-day-old rats, with
similar effects on gasping by the nonselective 
-receptor antagonist
propranolol. Thus these investigators concluded that gasping
requires intact adrenal function and primarily involves -adrenergic
receptors. However, no significant differences were found in
-adrenergic receptor expression within the putative gasping centers
of infants who succumbed to SIDS (20).
An interesting analogy to intermittent hypoxia can be drawn with prenatal nicotine exposures. Continuous administration of nicotine via an osmotic pump to pregnant rats from day 6 of pregnancy was associated with decreased gasp duration and with reduced ability to autoresuscitate until postnatal age 5-6 days but not thereafter (4, 5, 21). The adverse effect of perinatal nicotine was further shown by its acute administration to neonatal piglets at doses leading to serum levels similar to those found in infants of smoking mothers (7). In these experiments, laryngeal stimulation not only induced more severe central apneas in nicotine-treated piglets but also was associated with reduced ventilatory compensation after induced apnea (7). Thus both prenatal and early postnatal exposure to nicotine lead to impaired protective responses of rat pups that may sustain life during exposure to asphyxia. It should be emphasized, however, that the potentially deleterious effects of prenatal nicotine on gasping have not been consistently found (23). Whether these discrepant findings reflect the differences in dosages used or in the susceptibility of particular rat substrains remains unclear.
Although environmental temperature can modify gasping duration and the degree of successful autoresuscitation (24), we cannot a priori ascribe a role to this particular factor by virtue of our strict standardization of environmental temperature during all of our experiments. Similarly, all animals were studied at the same postnatal age, such that it is unlikely that there were marked differences in their developmental stages so as to induce the relatively robust changes in gasping and autoresuscitation characteristics associated with increasing postnatal age (6, 14, 18).
In summary, perinatal intermittent hypoxic exposures are associated with altered defense mechanisms against asphyxia, manifesting both as decreased total survival and as a relative inability to autoresuscitate. Although the implications of such increased susceptibility are unknown, we postulate that it may play a role in conditions such as SIDS.
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ACKNOWLEDGEMENTS |
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D. Gozal and E. Gozal are supported by National Institutes of Health Grants P20 RR-15576, HL-65270, HL-63912, and HL-66358; The Commonwealth of Kentucky Research Challenge Trust Fund; and American Heart Association Grant AHA-0050442N. S. R. Reeves was the recipient of a stipend from the University of Louisville Summer Research Scholar Program.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. Gozal, Kosair Children's Hospital Research Institute, Dept. of Pediatrics, Univ. of Louisville School of Medicine, 570 S. Preston St., Suite 321, Louisville, KY 40202-3830 (E-mail: david.gozal{at}louisville.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/japplphysiol.00972.2001
Received 25 September 2001; accepted in final form 1 November 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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