INTRODUCTION

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THE SUDDEN INFANT DEATH SYNDROME (SIDS) is defined as "the sudden death of an infant under 1 yr of age which remains unexplained after a thorough case investigation, including performance of a complete autopsy, examination of the death scene, and review of the clinical history" (44). In North America, SIDS is a leading cause of death beyond the neonatal period during the first year of life. SIDS is thought to occur during sleep or during the transition from sleep to wakefulness, and it occurs primarily between 2 wk and 8 mo of age, with 90% of SIDS cases occurring in the first 6 mo of life (44). Numerous epidemiological studies indicate that smoking during pregnancy is a major and independent risk factor for SIDS (3, 14, 15, 25, 26, 28, 29, 32, 33); this risk increases in proportion to the number of cigarettes smoked (14). In the National Institute of Child Health and Human Development study of epidemiological factors for SIDS, the relative risk for SIDS associated with maternal smoking was 3.4; this was higher than any other maternal or newborn condition evaluated, with a frequency of smoking among SIDS mothers of 70% (15). It is not known, however, how maternal smoking places offspring at an increased risk for SIDS.

Cigarette smoke contains a wide variety of chemicals, including nicotine (43). Nicotine is a neuroteratogen that easily crosses the placenta and is found in fetal cord blood in concentrations equal to or greater than those in maternal blood (23, 24, 39). Nicotine acts through highly selective and sensitive nicotinic cholinergic receptors and has the potential to influence the maturation of these receptors in fetal brain, autonomic ganglia, and the adrenal medulla (22, 34). Kinney et al. (19) have recently reported that human fetuses at midgestation have a heavy concentration of [³H]nicotine-binding sites in tegmental nuclei which govern cardiorespiratory integration, arousal, attention, rapid-eye-movement sleep, and somatic motor control. These [³H]nicotine-binding sites were found to decrease by 60-70% over the last half of gestation, leading the authors to suggest that mid-to-late gestation is a period when this region is most vulnerable to the harmful effects of nicotine from maternal smoking.

An inability to recover from prolonged sleep apnea has long been postulated as a possible factor in SIDS (12, 16, 17). In humans, recovery from sleep apnea is thought to occur early, as a result of arousal from sleep, or later, as a result of hypoxic gasping, when it is termed autoresuscitation (12, 41). Considering this and the aforementioned information on nicotine, one possible consequence of perinatal exposure to nicotine may be an alteration in protective responses that prevent severe hypoxia and death during prolonged sleep apnea. Our present experiments have been carried out to test the hypothesis that perinatal exposure to nicotine impairs the ability of newborn rats to autoresuscitate from primary apnea during repeated exposure to hypoxia, such as may occur during episodes of sleep apnea.

	METHODS

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Thirty-two Sprague-Dawley rat pups (5-6 days old) were studied. Each pup was born by spontaneous vaginal delivery and was housed with its mother and siblings (22 ± 1°C, 20-30% relative humidity, and 12:12-h light-dark cycle). Although 22°C is below the thermoneutral zone of newborn rats, each pup had the opportunity to select its ambient temperature between experiments by huddling with its siblings and/or mother (i.e., behavioral thermoregulation).

Surgical Preparation

All surgical and experimental procedures were carried out in accordance with the Guide to the Care and Use of Experimental Animals provided by the Canadian Council on Animal Care and with the approval of the Animal Care Committee of the University of Calgary.

Experimental Protocols

Experiment 1: Time to last gasp. For an experiment, each 5- to 6-day-old pup was removed from its mother and siblings, weighed, and placed in a metabolic chamber regulated at 37°C into which flowed room air at a rate of 1 l/min. At the end of a 30-min stabilization period, the gas that flowed into the chamber was changed from room air to 97% N₂-3% CO₂, and the time to last gasp was determined. At the beginning of the hypoxic exposure, the chamber was flushed with the hypoxic gas mixture until the gas concentrations in the chamber had stabilized; the flow rate was then lowered to 1 l/min. During a time-to-last-gasp experiment, the stages of the respiratory response to hypoxia as well as the time to last gasp were directly observed on the polygraph tracing. Eight pups of mothers that had received nicotine and eight pups of mothers that had received vehicle were studied.

Experiment 2. Autoresuscitation. For an experiment, each 5- to 6-day-old pup was removed from its mother and siblings, weighed, and placed into a metabolic chamber regulated at 37°C into which flowed room air at a rate of 1 l/min. At the end of a 30-min stabilization period, the gas that flowed into the metabolic chamber was changed from room air to 97% N₂-3% CO₂ until primary apnea occurred. The gas was then changed back to room air, and the ability of the pup to autoresuscitate by gasping was determined. This procedure was repeated at 5-min intervals until death occurred. Again, when the gas mixture was changed, the flow rate was increased until the gas concentrations in the chamber had stabilized; the flow rate was then lowered to 1 l/min. During an autoresuscitation experiment, primary apnea was detected by directly observing respiratory movements on the polygraph tracing. Autoresuscitation was deemed to occur when heart rate and respiratory rate returned to >60% of control levels within 5 min. Eight pups of mothers that had received nicotine and eight pups of mothers that had received vehicle were studied.

Experimental Apparatus

Experimental Measurements and Calculations

Nicotine

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1

Statistical Analysis

	RESULTS

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Experiment 1: Time to Last Gasp

View larger version (K): [in this window] [in a new window]   Fig. 1.   Three segments of a polygraph tracing showing respiratory response of 5-day-old rat pup to a single period of hypoxia. Variables shown are chamber CO2 concentration, electrocardiogram, and respiratory pattern. Exposure to a single period of hypoxia resulted in a reproducible respiratory response that consisted of an initial period of hyperpnea (1a) and arousal (1b) that preceded primary apnea (1c). Primary apnea was followed by a period of rapid gasping that lasted 1-2 min (1d). This period of rapid gasping was followed by a period of slower gasping of 1 or 2 gasps/min that lasted for 6-8 min (1e). Finally, there was a period of rapid gasping, which eventually waned and gave way to terminal apnea and death (1f ).

View larger version (K): [in this window] [in a new window]   Fig. 2.   Lack of effect of perinatal exposure to nicotine on heart rate response to a single period of hypoxia. Raw data and means ± SD are shown for each group of animals; n = 8 rat pups for each group.

View larger version (K): [in this window] [in a new window]   Fig. 3.   Lack of an effect of perinatal exposure to nicotine on gasping rate response to a single period of hypoxia. Raw data and means ± SD are shown for each group of animals; n = 8 rat pups in each group.

Experiment 2: Autoresuscitation

View larger version (K): [in this window] [in a new window]   Fig. 4.   Continuous polygraph tracing from a 5-day-old rat pup showing a successful autoresuscitation from primary apnea. During exposure to hypoxia, an initial period of hyperpnea (4a) and arousal (4b) preceded primary apnea and bradycardia (4c). Gasping was followed by an increase in heart rate (4d) and then restoration of normal respiratory pattern (4e).

View larger version (K): [in this window] [in a new window]   Fig. 5.   Lack of effect of perinatal exposure to nicotine on normoxemic heart rates after repeated exposure to hypoxia. Bars show means ± SD for each group of animals; n = 8 rat pups in each group.