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The cardiorespiratory response to anoxia: normal development and the effect of nicotine
Respiration Physiology 109 (1997) 231 – 239
The cardiorespiratory response to anoxia: normal development and the effect of nicotine J.N. Schuen a, O.S. Bamford b,*, J.L. Carroll b b
a DeVos Children’s Hospital at Butterworth, 100 Michigan St., NE, Grand Rapids, MI 49503, USA Eudowood Di6ision of Pediatric Respiratory Sciences, The Johns Hopkins Children’s Center, Park 316, 600 N. Wolfe St, Baltimore, MD 21287, USA
Received 10 July 1997; accepted 15 July 1997
Abstract Maternal smoking increases the risk of the sudden infant death syndrome (SIDS) 2 – 4-fold. The mechanism is unknown but may be related to hypoxia responses. Recovery from hypoxic apnea by young mammals depends on gasping and bradycardia. We asked whether prenatal nicotine exposure, reported to reduce hypoxic survival in 2 day old rat pups, acted by impairing gasping or bradycardia. Pregnant rats were infused throughout gestation and 1 week postnatally with nicotine tartrate (NIC) 12 mg/kg per day or saline (CON). Maternal plasma nicotine was 134.4 942 ng/ml, significantly reducing pup body weight. Pups at 3 – 28 days were exposed to anoxia (97% N2/3% CO2) until gasping ceased, while breathing and heart rate were recorded. NIC and CON groups were not significantly different at any age, in baseline heart rate, respiratory rate, the time course for bradycardia, time to gasp onset, duration of gasping, or number of gasps, although most of these variables declined significantly with age. We conclude that responses to anoxia are not affected by prenatal high-dose nicotine. © 1997 Elsevier Science B.V. Keywords: Control of breathing, hypoxia; Development; Hypoxia; Hypoxia, prenatal nicotine exposure; Mammals, rat; Pattern of breathing, gasping; Sudden infant death syndrome, prenatal nicotine exposure; Toxic agents, nicotine
1. Introduction Infant mammals, exposed to prolonged anoxia, become apneic and then begin gasping, with slow breaths at 3–4 times resting tidal volume and * Corresponding author. Tel.: +1 410 9555637; fax: + 1 410 9551030; e-mail: [email protected]
profound bradycardia. It is not clear whether bradycardia is reflex or due to direct action of hypoxia on the myocardium, or a combination of both, but its survival value is clear. The metabolic rate of young mammals is reduced in hypoxia (Mortola et al., 1989) and probably also in anoxia. Survival time is limited by the energy stores of the heart muscle (Mott, 1961) so that the
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combination of hypometabolism and bradycardia helps to prolong survival. Apnea reduces the metabolic demand of the respiratory muscles, while continuation of deep but infrequent gasping movements ensures that if oxygen becomes available, the PaO2 can rise to the point where eupnea can resume. Thus apnea and bradycardia prolong survival in severe hypoxia. Gasping is essential for re-oxygenation and autoresuscitation (Gershan et al., 1990), so that factors which impair the gasp response may decrease the chance of surviving episodes of severe hypoxia. Our hypothesis was that prenatal nicotine exposure would impair gasping. This hypothesis was an attempt to account for the observation that infants of smoking mothers have a 2 – 4 times increased risk of dying from SIDS compared with matched control infants (Schoendorf and Kiely, 1992; DiFranza and Lew, 1995; Taylor and Sanderson, 1995). Although this connection has been recognized, at least since 1988, the underlying mechanism is not understood. Cigarette smoke contains a wide range of neuroactive compounds of which nicotine and CO are present at the highest concentrations. While CO certainly affects O2 transport and hence may induce fetal hypoxia, direct effects of CO on fetal development appear to be minor (Koos et al., 1988; Lynch and Bruce, 1989). In contrast, nicotine is known to have multiple effects on development of the central nervous system: for example, impaired development of central cholinergic receptor sensitivity (Slotkin et al., 1991), striatal dopamine D2 binding (Janson et al., 1992) and catecholamine turnover in the cerebral cortex (Navarro et al., 1988) have been reported. It was therefore plausible that smoking might increase the SIDS risk by actions of nicotine on the control of breathing. We have recently shown that prenatal nicotine exposure has no significant effect on the control of eupnea in developing rats (Bamford et al., 1996). However, there is abundant evidence that gasping and eupnea are controlled independently (St. John, 1990) and the possibility therefore remained that nicotine might impair the ability to autoresuscitate through gasping. Autoresuscitation is the process whereby infant mammals in hypoxic apnea can recover sponta-
neously if oxygen becomes available. We therefore examined the effects of prenatal chronic exposure to nicotine on the ability to gasp in anoxia, and on the time course over which resistance to anoxia changes postnatally.
2. Methods Pregnant Sprague Dawley rats (CD rats, Charles River) were delivered to the animal facility on day 2 of gestation, housed individually throughout gestation and given food and water ad lib. Under general anesthesia (Metofane inhalation), pregnant rats underwent subcutaneous placement of an osmotic minipump (type 2ML4: Alza) on day 2 or 3 of gestation. The pump delivered either normal sterile saline (0.9% NaCl) or 12 mg/kg per day nicotine bitartrate for 4 weeks. Nicotine was made up in sterile saline (0.9% NaCl). The pump delivered 70 ml/day and the nicotine solution was made up to deliver the calculated dose rate, based on the average expected body weight throughout gestation. Maternal blood was obtained for nicotine levels (National Medical Services, Willow Grove, PA.) from tail vein samples. Ventilation was recorded by flow plethysmography, using methods based on those of Mortola et al. (1989) and Eden and Hanson (1987). Breathing displaces air in the plethysmograph, setting up a tidal flow which is measured using a pneumotachometer and then integrated to give tidal volume. Flow, volume, EKG signal and heart rate were recorded on paper (Gould ES2000). Calibration of volume recordings was accomplished by injecting known volumes of air into the plethysmography chamber. The system had a linear response up to twice the maximum flow rate produced by the rat pups. To minimize variations in ventilatory and cardiac responses, recordings were taken from quiet, unanesthetized rat pups at a constant temperature of 32°C. Gas mixtures were delivered dry and at room temperature. Responses to anoxia were measured in rat pups in the following age ranges: 2–4, 6–8, 12–14, 20–22, and 26–28 days old. Weaning was at 21–23 days. Pups were weighed after rectal or
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oral temperatures were recorded. Wire electrodes (Teflon-insulated, 70 m) for EKG recording were placed subcutaneously using 25 g hypodermic needles and then the rat pup was secured in the plethysmography chamber. The neck made a gastight fit in a hole in the taut rubber membrane that formed the front of the plethysmograph. Air at 11/min supplied the anterior chamber which fit loosely over the rat’s head. The pup was allowed to acclimate for 5–10 min to the plethysmography chamber and was then exposed to anoxia by passing 97% nitrogen and 3% carbon dioxide into the anterior chamber. PO2 and PCO2 of outflow gas from the chamber was monitored (S3A-II and CD3A gas analyzers, Ametek). The onset of anoxia was followed within a few breaths by apnea and apparent unconsciousness, with no response to cutaneous stimulation. Slow stereotyped gasping started after 30 – 90 sec. The experiment was terminated when gasping ceased and the rat was sacrificed by an overdose of methoxyflurane anesthesia. For each anoxic trial, gasp duration, gasp frequency and gasp interval were determined, as previously defined (Gershan et al., 1992). We also measured: time to first gasp (TTFG; the time from the beginning of anoxia to the first gasp); time to last gasp (TTLG; time from the initiation of anoxia to the time where gasp volumes fell to less than 10% of initial recorded gasps) and the number of gasps per trial (total number of gasps between the first and last significant gasp). Gasp volume was not measured as it was found that movement artifact from the arching associated with gasping made volume measurements inaccurate. All procedures were approved by the Institutional Animal Care and Use Committee.
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rately. Homogeneity of variance was tested (Bartlett test). Post hoc testing with the NewmanKeuls comparison determined which groups within ANOVA were significantly different. All values were expressed as mean9 SD. PB 0.05 was considered significant.
3. Results A total of 27 pregnant rats (13 nicotine, 14 control) had pumps implanted. There were 276 live pups. There were no maternal deaths and no significant complications related to the osmotic mini-pumps. A total of 172 pups, 91 control and 81 nicotine, were used in the anoxia experiments. The mean litter sizes were 11.4 9 2.7 in the nicotine group and 11.69 2.0 in the control population (n.s.). A total of 11 stillbirths were noted in three nicotine treated litters and none in the control group. There were no other post-natal deaths or evidence of malformations in the offspring. Mean nicotine level during gestation was 1349 42 g (n= 10) and the post-gestational mean was 69.59 12.6 g. Nicotine pups weighed less at all ages, but the differences were statistically significant only at birth (PB0.0001), and at 12–14 and
2.1. Statistical analysis Our study examined both the development of cardiorespiratory responses to anoxia and the effect of nicotine on this development. Analysis of variance (ANOVA) with simple main effects was employed to examine the effects of two independent variables (i.e. different ages of the pups, treatment groups) with each dependent variable (e.g. weight, TTFG, TTLG, etc.) studied sepa-
Fig. 1. Effect of chronic nicotine exposure on body weight in rat pups. Weight is shown as mean9 SD. Control group rats were consistently heavier than chronically nicotine-exposed rats at all ages, the difference reaching statistical significance at three ages (shown by asterisks). Data were obtained from variable number of rats at different ages: n is shown above each bar.
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Fig. 3. Effect of nicotine exposure and age on the time to first gasp (TTFG) when rat pups are exposed to anoxia. TTFG declined with age in both NIC and CON groups and was consistently slightly higher in CON but the difference did not reach statistical significance at any age.
Fig. 2. Breathing and heart rate before and during exposure to anoxia in a 3-day rat pup. (a) shows breathing and EKG under baseline conditions. (b) shows apnea 5 min after the onset of anoxia. Fig. 2c shows an anoxic gasp one min later. Vertical and horizontal scales apply to all three records. Each record lasts 10 sec.
at 26–28 days (PB 0.05). (Fig. 1). Baseline heart rate, respiratory rate, minute ventilation and body temperature were not significantly different between nicotine and control groups at any age. Temperature was not recorded during the experiments. Breathing 97% nitrogen and 3% CO2 induced apnea within a few sec. Apnea lasted from 82 sec at 3 days to 18 sec at 28 days. Following the brief apneic period, all rats gasped regularly. There were no gasp failures in any litter at any age. Fig. 2. shows representative recordings from an anoxia experiment in a 7 day old pup. Breathing and EKG are shown for baseline (Fig. 2a), at 50 sec (Fig. 2b) and 13 min (Fig. 2c). The heart rate fell from 420 to 114 bpm during the period of
anoxic apnea. Apnea was followed by gasping, during which the heart rate reached a constant 75–80 bpm. This pattern was maintained until gasping ceased. The delay between the onset of anoxia and the first gasp (TTFG, Fig. 3) fell from about 80 sec at 3 days to 30 sec at 28 days. TTFG was consistently slightly longer in the CON group, but the difference was not statistically significant at any age. The time from anoxia onset to the last signifi-
Fig. 4. Effect of nicotine exposure and age on the time to last gasp (TTLG) in anoxia. TTLG declined significantly with age in both groups but there was no significant difference between groups.
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Fig. 5. Decline of heart rate plotted against time in anoxia. At all ages, heart rate fell precipitously at the onset of anoxia and then reached a level which was more or less sustained until after the last gasp. This steady heart rate was lowest in the 3 day old pups and increased with age. There was no difference between NIC and CON groups in the time course of the heart rate response at any age.
cant gasp (TTLG, Fig. 4) was 38 – 40 min at 3 days, falling to less than 20 min by 6 days and less than 5 min at 20 days. NIC and CON groups were not significantly different at any age. Baseline heart rates were between 400 and 500/ min except in the 3-day group where the baseline was under 300/min. Heart rate variability was not measured. Immediately after the onset of anoxia the EKG signal became uninterpretable due to movement artifact. Within 10 – 15 sec after the onset of anoxia, the heart rate fell to approximately 1/4 to 1/6 of the baseline heart rate and
thereafter remained almost constant throughout the period of gasping, always continuing after the last gasp. In no case did the terminal decline in heart rate precede the end of gasping. The final heart rate reached during anoxic gasping was lowest in the younger pups. At 3 and 7 days a steady heart rate of less than 60/min was sustained until after gasping ceased. At 14 days the lowest rate observed was about 80/min while at older ages, heart rate did not fall below 100/min until the final decline. There were no significant differences between the two treatment groups at
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any age (Fig. 5). There was no evidence of cardiac arrhythmia or heart block during the pup’s gasping efforts, however, the amplitude of the QRS wave was reduced towards the period of gasping.
4. Discussion
4.1. Main findings This study was intended to determine the developmental profile of the gasping response to anoxia in rat pups and to measure any effect of chronic nicotine exposure on the profile. The main new finding from this study is that chronic prenatal nicotine exposure, at doses which significantly reduced body weight, had no effect on the development of either anoxic gasping or bradycardia at any age. In addition we confirm that the main developmental characteristics of the response to anoxia can be summarized as follows: (a) the longest observed gasping duration was at 3 days. Duration and number of gasps declined in older rats; (b) anoxic gasping occurs at all ages up to 28 days; (c) the gasp rate is almost constant until death in the 3 day old rats but in older rats, gasping accelerates during the anoxic exposure; (d) regular QRS complexes continue long after anoxic gasping ceases; and (e) the heart rate falls rapidly at the onset of anoxia and then remains fairly stable until after gasping ceases. This stable, anoxic value for heart rate increased with age.
4.2. Significance of gasping Gasping is initiated and controlled by a mechanism that is anatomically and functionally distinct from the control of eupnea (St. John, 1990). Gasping and bradycardia are part of the autoresuscitation process, an important defense mechanism. (Gershan et al., 1990; Jacobi and Thach, 1989). Extreme hypoxia or anoxia rapidly induces apnea, which is then followed after a period varying from a few sec to a min by deep inspiratory gasps. Hypoxic hypometabolism probably also occurs (Mortola and Rezzonico, 1988; Mortola et al., 1989) Between gasps there is usually no movement, with low heart rate and little variability.
Gasps are associated with transient acceleration of heart rate. It is not clear whether the gasp-related acceleration is due to pulmonary stretch reflexes or is a direct effect of increased venous return. Chemoreceptor stimulation is not a factor in gasp-related cardiac acceleration, as acceleration occurs with gasps in an anoxic atmosphere. If a gasping rat is supplied with room air, the next gasp is soon followed by a rise in heart rate and a resumption of eupneic breathing (autoresuscitation) then occurs (Gershan et al., 1990). Gasping and bradycardia in response to anoxia/hypoxia appears to have two important benefits: apnea and bradycardia help in conserving oxygen, while the infrequent but intense inspiratory efforts can re-oxygenate the body if oxygen becomes available. Thus gasping forms part of a mechanism that allows infant mammals to survive periods of severe hypoxia. In infants, severe hypoxia may occur if for example the airway becomes obstructed by bedding and the infant fails to arouse. Gasping may then re-oxygenate the infant sufficiently for breathing to resume. Thus the ability to gasp, while normally unused, may become crucial for survival. Impaired ability to gasp would then reduce the infants chance of surviving a severe hypoxic challenge.
4.3. Rationale for methods In order to test our hypothesis, we induced gasping by anoxia. While complete anoxia is unlikely to occur naturally, the intention was not to model a real-life situation but simply to induce gasping rapidly and consistently. Apnea and gasping have occasionally been observed during severe hypoxia in our laboratory. Gasping is identical in hypoxia and anoxia, but analysis in hypoxia is complicated by the fact that the hypoxic animal can then re-oxygenate and resume eupneic breathing. For these reasons we used anoxia as a challenge. The study was not designed to measure survival time and no autoresuscitation was allowed. We have no direct evidence that the gasping animals would in fact have survived, as none of the animals was allowed to re-oxygenate. However, pub-
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lished data (Fazekas et al., 1941; Mott, 1961) and our own experience strongly suggest that survival is possible up to the last significant gasp and that the time to last gasp is therefore a measure of the survivable period of anoxia.
4.4. Confirmation of effecti6e dosing The main finding of this study was negative. No effect of nicotine on gasping occurred at any age. The question therefore arises whether the nicotine reached the fetuses in an effective dose. Mean maternal plasma nicotine during gestation was 134 ng/ml, compared with the range of 20 – 40 ng/ml found in human smokers (Lichtensteiger et al., 1988). Nicotine crosses the placenta readily and so there is every reason to believe the developing fetuses were exposed to nicotine: some studies have shown higher levels of nicotine in fetal than maternal plasma (Ruhle et al., 1995). Dose rates as low as 2 mg/kg per day have been shown to affect the development of cholinergic receptors in the CNS (Zahalka et al., 1992). Moreover, the nicotine-exposed rats were consistently lighter than control. This evidence strongly suggest that the fetal rats were exposed to sufficient nicotine to have significant physiological effects.
4.5. Factors influencing gasping time Our data show a marked decline in gasping time with age. This is consistent with previous published data that show survival times of 30 – 50 min in newborns, falling to less than 10 min by 2 weeks (Stafford and Weatherall, 1960; Mott, 1961). Neither the specific cause of gasping cessation, nor the decline in gasping time with age have been explained. The termination of gasping may be due to accumulation of neurotoxic amino acids. Valproate, which reduces the levels of aspartate and glutamate in the brains of mice, increases their survival time in anoxia (Thurston and Hauhart, 1989). However, most of the literature supports the idea that gasping ceases when energy substrates are exhausted, so that gasping stops earlier if metabolic rate is increased or if substrate stores are reduced. For example, Mott (1961) reported a linear relation between survival
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time and cardiac carbohydrate content in several species. Substrate exhaustion in respiratory muscle may also account for the postnatal decline in gasping time: a parallel postnatal decline in glycogen content of skeletal muscle and heart has been demonstrated (Shelley, 1961). More recently it has been shown that survival time of rats in severe hypoxia is directly related to their tissue glycogen content (Purshottam et al., 1978) and that diurnal variation in hypoxic survival is related to diurnal changes in the ability to mobilize liver glycogen (Kwarecki et al., 1984). Increasing the cerebral metabolic rate with aminophylline reduces gasp time in severed mouse heads (Thurston et al., 1978). Prolonged nicotine exposure has been shown to increase sympathetic nervous system activation of brown fat thermogenesis (Lupien and Bray, 1988). Although metabolic rate was not measured in these studies, thermogenesis is associated with increased metabolism, so that it might be expected that metabolic substrates would be exhausted earlier in nicotine-treated anoxic animals. Our finding that QRS complexes continued after the end of gasping agrees with previous studies (Stafford and Weatherall, 1960; Adolph, 1969). This observation appears to be inconsistent with the finding by Mott (1961) that survival time was related to energy substrates in the heart. However, QRS complexes may not be associated with significant contraction and stroke volume in conditions of extreme acidosis and tissue hypoxia, such as occur in our experiments, and we cannot be certain that cerebral perfusion persists in anoxia. Thus survival time may be limited by cardiac contractility, although electrical activity continues after gasping has ceased. Heart rate variability was not measured but appeared from a heart rate display to be very low during anoxia in both nicotine and control groups. If true, this would imply a low level of cardiac sympathetic activity under these conditions, as might be expected with an anoxic brainstem. Under these conditions the heart may receive essentially no neural input and be functionally denervated. However, we have no data on the neural control of the heart in anoxia. The specific system failure that causes cessation of gasping and heart beat in anoxia remains un-
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known, but we can safely conclude that it is unaffected by nicotine exposure.
5. Discrepancies with other literature There are some interesting discrepancies between our findings and some previous studies. All of our rats, at all ages, gasped in anoxia, with the youngest rats surviving for the longest time. This is in contrast to mice in which gasping ability showed a minimum at 17 – 23 days (Jacobi and Thach, 1989). Most other accounts of anoxic gasping, e.g. (Fazekas et al., 1941; Stafford and Weatherall, 1960) report that gasping time falls exponentially from newborn to adult and the reason for the anomalous responses of mice is unclear. No arrhythmias were observed in our studies. Bradycardia was established at the onset of anoxia and a low but steady heart rate persisted until after gasping ceased. As we did not attempt resuscitation we do not know if heart block would have occurred during re-oxygenation, but there was no evidence of heart block during gasping as has been reported (Gershan et al., 1992). Differences between strains of mice in their susceptibility to anoxic heart block have been reported (Gershan et al., 1992). Adolph (1969) noted that some ground squirrels restored breathing after anoxic apnea but failed to return to a normal heart rate and died: presumably these animals were in heart block, but no details are given. The apparent discrepancy between these findings and our study may have occurred because we did not attempt resuscitation. Gershan et al. (1992) reported that the probability of heart block in the less susceptible strain of mice increased with successive anoxic challenges, and Adolph (1969) observed it only during recovery. Thus, discrepancies between our studies and those of other workers are probably partly due to different protocols and partly indications of species differences. The physiological significance of such inter- and intra-specific differences, and their cause, is unclear. Adolph (1969) reported wide differences between species in both survival time in nitrogen and in the time course of its postnatal
change, with 50% survival for 10 min at ages ranging from 3 to 16 days. Tolerance to anoxia at birth was highest in the most immature newborns and the postnatal loss of tolerance occurred most rapidly in the fastest developing species. Clearly the loss of anoxia tolerance is part of a developmental process, but in rats it appears to be unaffected by nicotine exposure. In conclusion, prenatal exposure to nicotine, at doses that cause pre- and postnatal growth retardation, does not affect anoxic gasping or bradycardia in the rat pup at any age between 3 and 28 days. This finding is not consistent with our hypothesis that the increased risk of SIDS in infants of smoking mothers is mediated through an action of nicotine on the ability to survive and recover from extreme hypoxia. The mechanism by which maternal smoking increases the risk of SIDS remains unknown.
Acknowledgements Dr Schuen was supported by a Cystic Fibrosis Foundation training grant B856 (1994–1995) and an American Lung Association fellowship grant. Dr Carroll was supported by NIH HL02543-01. Dr Bamford was supported by a research grant from the SIDS Alliance (SP 0017).
References Adolph, E.F., 1969. Regulations during survival without oxygen in infant mammals. Respir. Physiol. 7, 356 – 368. Bamford, O.S., Schuen, J.N., Carroll, J.L., 1996. Effect of nicotine exposure on postnatal ventilatory responses to hypoxia and hypercapnia. Respir. Physiol. 106, 1 – 11. DiFranza, J.R., Lew, R.A., 1995. Effect of maternal cigarette smoking on pregnancy complications and sudden infant death syndrome. J. Fam. Pract. 40, 385 – 394. Eden, G.J., Hanson, M.A., 1987. Maturation of the respiratory response to acute hypoxia in the newborn rat. J. Physiol. (London) 392, 1 – 9. Fazekas, J.F., Alexander, F.A.D., Himwich, H.E., 1941. Tolerance of the newborn to anoxia. Am. J. Physiol. 134, 281 – 287. Gershan, W.M., Jacobi, M.S., Thach, B.T., 1990. Maturation of cardiorespiratory interactions in spontaneous recovery from hypoxic apnea (autoresuscitation). Pediatr. Res. 28, 87 – 93.
J.N. Schuen et al. / Respiration Physiology 109 (1997) 231–239 Gershan, W.M., Jacobi, M.S., Thach, B.T., 1992. Mechanisms underlying induced autoresuscitation failure in BALB/c and SWR mice. J. Appl. Physiol. 72, 677–685. Jacobi, M.S., Thach, B.T., 1989. Effect of maturation on spontaneous recovery from hypoxic apnea by gasping. J. Appl. Physiol. 66, 2384–2390. Janson, A.M., Hedlund, P.B., Hillefors, M., von-Euler, G., 1992. Chronic nicotine treatment decreases dopamine D2 agonist binding in the rat basal ganglia. Neuroreport 3, 1117 – 1120. Koos, B.J., Matsuda, K., Power, G.G., 1988. Fetal breathing and sleep state responses to graded carboxyhemoglobinemia in sheep. J. Appl. Physiol. 65, 2118–2123. Kwarecki, K., Debiec, H., Wroblewski, S., 1984. Biological time-related changes in tolerance of male rats to hypoxia– I. Survival rate and carbohydrate metabolism. Chronobiol. Int. 1, 239 – 244. Lichtensteiger, W., Ribary, U., Schlumpf, M., Odermatt, B., Widmer, H.R., 1988. Prenatal adverse effects of nicotine on the developing brain. Prog. Brain. Res. 73, 137–157. Lupien, J.R., Bray, G.A., 1988. Nicotine increases thermogenesis in brown adipose tissue in rats. Pharmacol. Biochem. Behav. 29, 33 – 37. Lynch, A.M., Bruce, N.W., 1989. Placental growth in rats exposed to carbon monoxide at selected stages of pregnancy. Biol. Neonate 56, 151–157. Mortola, J.P., Rezzonico, R., 1988. Metabolic and ventilatory rates in newborn kittens during acute hypoxia. Respir. Physiol. 73, 55 – 67. Mortola, J.P., Rezzonico, R., Lanthier, C., 1989. Ventilation and oxygen consumption during acute hypoxia in newborn mammals. A comparative analysis. Respir. Physiol. 78, 31– 43. Mott, J.C., 1961. Ability of young mammals to withstand total oxygen lack. Br. Med. Bull. 17, 144–148. Navarro, H.A., Seidler, F.J., Whitmore, W.L., Slotkin, T.A., 1988. Prenatal exposure to nicotine via maternal infusions. Effects on development of catecholamine systems. J. Pharmacol. Exp. Ther. 244, 940–944.
.
239
Purshottam, T., Kaveeshwar, U., Brahmachari, H.D., 1978. Hypoxia tolerance in rats in relation to tissue glycogen levels. Aviat. Space Environ. Med. 49, 1062 – 1064. Ruhle, W., Graf von Ballestrem, C.L., Pult, H.M., Gnirs, J., 1995. Correlation of cotinine level in amniotic fluid, umbilical artery blood and maternal blood. Geburtshilfe Frauenheilkd. 55, 156 – 159. Schoendorf, K.C., Kiely, J.L., 1992. Relationship of sudden infant death syndrome to maternal smoking during and after pregnancy. Pediatrics 90, 905 – 908. Shelley, H.J., 1961. Glycogen reserves and their changes at birth. Br. Med. Bull. 17, 137 – 143. Slotkin, T.A., Lappi, S.E., Tayyeb, M.I., Seidler, F.J., 1991. Chronic prenatal nicotine exposure sensitizes rat brain to acute postnatal nicotine challenge as assessed with ornithine decarboxylase. Life Sci. 49, 665 – 670. Stafford, A., Weatherall, J.A.C., 1960. The survival of young rats in nitrogen. J. Physiol. (London) 153, 457 – 472. St. John, W., 1990. Neurogenesis, control and functional significance of gasping. J. Appl. Physiol. 68, 1305 – 1315. Taylor, J.A., Sanderson, M., 1995. A reexamination of the risk factors for the sudden infant death syndrome. J. Pediatr. 126, 887 – 891. Thurston, J.H., Hauhard, R.E., Dirgo, J.A., 1978. Aminophylline increases cerebral metabolic rate and decreases anoxic survival in young mice. Science 201, 649 – 651. Thurston, J.H., Hauhart, R.E., 1989. Valproate doubles the anoxic survival time of normal developing mice. Possible relevance to valproate-induced decreases in cerebral levels of glutamate and aspartate and increases in taurine. Life Sci. 45, 59 – 62. Zahalka, E.A., Seidler, F.J., Lappi, S.E., McCook, E.C., Yanai, J., Slotkin, T.A., 1992. Deficits in development of central cholinergic pathways caused by fetal nicotine exposure. Differential effects on choline acetyltransferase activity and [3H]hemicholinium-3 binding. Neurotoxicol. Teratol. 14, 375 – 382.
The cardiorespiratory response to anoxia: normal development and the effect of nicotine J.N. Schuen a, O.S. Bamford b,*, J.L. Carroll b b
a DeVos Children’s Hospital at Butterworth, 100 Michigan St., NE, Grand Rapids, MI 49503, USA Eudowood Di6ision of Pediatric Respiratory Sciences, The Johns Hopkins Children’s Center, Park 316, 600 N. Wolfe St, Baltimore, MD 21287, USA
Received 10 July 1997; accepted 15 July 1997
Abstract Maternal smoking increases the risk of the sudden infant death syndrome (SIDS) 2 – 4-fold. The mechanism is unknown but may be related to hypoxia responses. Recovery from hypoxic apnea by young mammals depends on gasping and bradycardia. We asked whether prenatal nicotine exposure, reported to reduce hypoxic survival in 2 day old rat pups, acted by impairing gasping or bradycardia. Pregnant rats were infused throughout gestation and 1 week postnatally with nicotine tartrate (NIC) 12 mg/kg per day or saline (CON). Maternal plasma nicotine was 134.4 942 ng/ml, significantly reducing pup body weight. Pups at 3 – 28 days were exposed to anoxia (97% N2/3% CO2) until gasping ceased, while breathing and heart rate were recorded. NIC and CON groups were not significantly different at any age, in baseline heart rate, respiratory rate, the time course for bradycardia, time to gasp onset, duration of gasping, or number of gasps, although most of these variables declined significantly with age. We conclude that responses to anoxia are not affected by prenatal high-dose nicotine. © 1997 Elsevier Science B.V. Keywords: Control of breathing, hypoxia; Development; Hypoxia; Hypoxia, prenatal nicotine exposure; Mammals, rat; Pattern of breathing, gasping; Sudden infant death syndrome, prenatal nicotine exposure; Toxic agents, nicotine
1. Introduction Infant mammals, exposed to prolonged anoxia, become apneic and then begin gasping, with slow breaths at 3–4 times resting tidal volume and * Corresponding author. Tel.: +1 410 9555637; fax: + 1 410 9551030; e-mail: [email protected]
profound bradycardia. It is not clear whether bradycardia is reflex or due to direct action of hypoxia on the myocardium, or a combination of both, but its survival value is clear. The metabolic rate of young mammals is reduced in hypoxia (Mortola et al., 1989) and probably also in anoxia. Survival time is limited by the energy stores of the heart muscle (Mott, 1961) so that the
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combination of hypometabolism and bradycardia helps to prolong survival. Apnea reduces the metabolic demand of the respiratory muscles, while continuation of deep but infrequent gasping movements ensures that if oxygen becomes available, the PaO2 can rise to the point where eupnea can resume. Thus apnea and bradycardia prolong survival in severe hypoxia. Gasping is essential for re-oxygenation and autoresuscitation (Gershan et al., 1990), so that factors which impair the gasp response may decrease the chance of surviving episodes of severe hypoxia. Our hypothesis was that prenatal nicotine exposure would impair gasping. This hypothesis was an attempt to account for the observation that infants of smoking mothers have a 2 – 4 times increased risk of dying from SIDS compared with matched control infants (Schoendorf and Kiely, 1992; DiFranza and Lew, 1995; Taylor and Sanderson, 1995). Although this connection has been recognized, at least since 1988, the underlying mechanism is not understood. Cigarette smoke contains a wide range of neuroactive compounds of which nicotine and CO are present at the highest concentrations. While CO certainly affects O2 transport and hence may induce fetal hypoxia, direct effects of CO on fetal development appear to be minor (Koos et al., 1988; Lynch and Bruce, 1989). In contrast, nicotine is known to have multiple effects on development of the central nervous system: for example, impaired development of central cholinergic receptor sensitivity (Slotkin et al., 1991), striatal dopamine D2 binding (Janson et al., 1992) and catecholamine turnover in the cerebral cortex (Navarro et al., 1988) have been reported. It was therefore plausible that smoking might increase the SIDS risk by actions of nicotine on the control of breathing. We have recently shown that prenatal nicotine exposure has no significant effect on the control of eupnea in developing rats (Bamford et al., 1996). However, there is abundant evidence that gasping and eupnea are controlled independently (St. John, 1990) and the possibility therefore remained that nicotine might impair the ability to autoresuscitate through gasping. Autoresuscitation is the process whereby infant mammals in hypoxic apnea can recover sponta-
neously if oxygen becomes available. We therefore examined the effects of prenatal chronic exposure to nicotine on the ability to gasp in anoxia, and on the time course over which resistance to anoxia changes postnatally.
2. Methods Pregnant Sprague Dawley rats (CD rats, Charles River) were delivered to the animal facility on day 2 of gestation, housed individually throughout gestation and given food and water ad lib. Under general anesthesia (Metofane inhalation), pregnant rats underwent subcutaneous placement of an osmotic minipump (type 2ML4: Alza) on day 2 or 3 of gestation. The pump delivered either normal sterile saline (0.9% NaCl) or 12 mg/kg per day nicotine bitartrate for 4 weeks. Nicotine was made up in sterile saline (0.9% NaCl). The pump delivered 70 ml/day and the nicotine solution was made up to deliver the calculated dose rate, based on the average expected body weight throughout gestation. Maternal blood was obtained for nicotine levels (National Medical Services, Willow Grove, PA.) from tail vein samples. Ventilation was recorded by flow plethysmography, using methods based on those of Mortola et al. (1989) and Eden and Hanson (1987). Breathing displaces air in the plethysmograph, setting up a tidal flow which is measured using a pneumotachometer and then integrated to give tidal volume. Flow, volume, EKG signal and heart rate were recorded on paper (Gould ES2000). Calibration of volume recordings was accomplished by injecting known volumes of air into the plethysmography chamber. The system had a linear response up to twice the maximum flow rate produced by the rat pups. To minimize variations in ventilatory and cardiac responses, recordings were taken from quiet, unanesthetized rat pups at a constant temperature of 32°C. Gas mixtures were delivered dry and at room temperature. Responses to anoxia were measured in rat pups in the following age ranges: 2–4, 6–8, 12–14, 20–22, and 26–28 days old. Weaning was at 21–23 days. Pups were weighed after rectal or
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oral temperatures were recorded. Wire electrodes (Teflon-insulated, 70 m) for EKG recording were placed subcutaneously using 25 g hypodermic needles and then the rat pup was secured in the plethysmography chamber. The neck made a gastight fit in a hole in the taut rubber membrane that formed the front of the plethysmograph. Air at 11/min supplied the anterior chamber which fit loosely over the rat’s head. The pup was allowed to acclimate for 5–10 min to the plethysmography chamber and was then exposed to anoxia by passing 97% nitrogen and 3% carbon dioxide into the anterior chamber. PO2 and PCO2 of outflow gas from the chamber was monitored (S3A-II and CD3A gas analyzers, Ametek). The onset of anoxia was followed within a few breaths by apnea and apparent unconsciousness, with no response to cutaneous stimulation. Slow stereotyped gasping started after 30 – 90 sec. The experiment was terminated when gasping ceased and the rat was sacrificed by an overdose of methoxyflurane anesthesia. For each anoxic trial, gasp duration, gasp frequency and gasp interval were determined, as previously defined (Gershan et al., 1992). We also measured: time to first gasp (TTFG; the time from the beginning of anoxia to the first gasp); time to last gasp (TTLG; time from the initiation of anoxia to the time where gasp volumes fell to less than 10% of initial recorded gasps) and the number of gasps per trial (total number of gasps between the first and last significant gasp). Gasp volume was not measured as it was found that movement artifact from the arching associated with gasping made volume measurements inaccurate. All procedures were approved by the Institutional Animal Care and Use Committee.
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rately. Homogeneity of variance was tested (Bartlett test). Post hoc testing with the NewmanKeuls comparison determined which groups within ANOVA were significantly different. All values were expressed as mean9 SD. PB 0.05 was considered significant.
3. Results A total of 27 pregnant rats (13 nicotine, 14 control) had pumps implanted. There were 276 live pups. There were no maternal deaths and no significant complications related to the osmotic mini-pumps. A total of 172 pups, 91 control and 81 nicotine, were used in the anoxia experiments. The mean litter sizes were 11.4 9 2.7 in the nicotine group and 11.69 2.0 in the control population (n.s.). A total of 11 stillbirths were noted in three nicotine treated litters and none in the control group. There were no other post-natal deaths or evidence of malformations in the offspring. Mean nicotine level during gestation was 1349 42 g (n= 10) and the post-gestational mean was 69.59 12.6 g. Nicotine pups weighed less at all ages, but the differences were statistically significant only at birth (PB0.0001), and at 12–14 and
2.1. Statistical analysis Our study examined both the development of cardiorespiratory responses to anoxia and the effect of nicotine on this development. Analysis of variance (ANOVA) with simple main effects was employed to examine the effects of two independent variables (i.e. different ages of the pups, treatment groups) with each dependent variable (e.g. weight, TTFG, TTLG, etc.) studied sepa-
Fig. 1. Effect of chronic nicotine exposure on body weight in rat pups. Weight is shown as mean9 SD. Control group rats were consistently heavier than chronically nicotine-exposed rats at all ages, the difference reaching statistical significance at three ages (shown by asterisks). Data were obtained from variable number of rats at different ages: n is shown above each bar.
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Fig. 3. Effect of nicotine exposure and age on the time to first gasp (TTFG) when rat pups are exposed to anoxia. TTFG declined with age in both NIC and CON groups and was consistently slightly higher in CON but the difference did not reach statistical significance at any age.
Fig. 2. Breathing and heart rate before and during exposure to anoxia in a 3-day rat pup. (a) shows breathing and EKG under baseline conditions. (b) shows apnea 5 min after the onset of anoxia. Fig. 2c shows an anoxic gasp one min later. Vertical and horizontal scales apply to all three records. Each record lasts 10 sec.
at 26–28 days (PB 0.05). (Fig. 1). Baseline heart rate, respiratory rate, minute ventilation and body temperature were not significantly different between nicotine and control groups at any age. Temperature was not recorded during the experiments. Breathing 97% nitrogen and 3% CO2 induced apnea within a few sec. Apnea lasted from 82 sec at 3 days to 18 sec at 28 days. Following the brief apneic period, all rats gasped regularly. There were no gasp failures in any litter at any age. Fig. 2. shows representative recordings from an anoxia experiment in a 7 day old pup. Breathing and EKG are shown for baseline (Fig. 2a), at 50 sec (Fig. 2b) and 13 min (Fig. 2c). The heart rate fell from 420 to 114 bpm during the period of
anoxic apnea. Apnea was followed by gasping, during which the heart rate reached a constant 75–80 bpm. This pattern was maintained until gasping ceased. The delay between the onset of anoxia and the first gasp (TTFG, Fig. 3) fell from about 80 sec at 3 days to 30 sec at 28 days. TTFG was consistently slightly longer in the CON group, but the difference was not statistically significant at any age. The time from anoxia onset to the last signifi-
Fig. 4. Effect of nicotine exposure and age on the time to last gasp (TTLG) in anoxia. TTLG declined significantly with age in both groups but there was no significant difference between groups.
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Fig. 5. Decline of heart rate plotted against time in anoxia. At all ages, heart rate fell precipitously at the onset of anoxia and then reached a level which was more or less sustained until after the last gasp. This steady heart rate was lowest in the 3 day old pups and increased with age. There was no difference between NIC and CON groups in the time course of the heart rate response at any age.
cant gasp (TTLG, Fig. 4) was 38 – 40 min at 3 days, falling to less than 20 min by 6 days and less than 5 min at 20 days. NIC and CON groups were not significantly different at any age. Baseline heart rates were between 400 and 500/ min except in the 3-day group where the baseline was under 300/min. Heart rate variability was not measured. Immediately after the onset of anoxia the EKG signal became uninterpretable due to movement artifact. Within 10 – 15 sec after the onset of anoxia, the heart rate fell to approximately 1/4 to 1/6 of the baseline heart rate and
thereafter remained almost constant throughout the period of gasping, always continuing after the last gasp. In no case did the terminal decline in heart rate precede the end of gasping. The final heart rate reached during anoxic gasping was lowest in the younger pups. At 3 and 7 days a steady heart rate of less than 60/min was sustained until after gasping ceased. At 14 days the lowest rate observed was about 80/min while at older ages, heart rate did not fall below 100/min until the final decline. There were no significant differences between the two treatment groups at
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any age (Fig. 5). There was no evidence of cardiac arrhythmia or heart block during the pup’s gasping efforts, however, the amplitude of the QRS wave was reduced towards the period of gasping.
4. Discussion
4.1. Main findings This study was intended to determine the developmental profile of the gasping response to anoxia in rat pups and to measure any effect of chronic nicotine exposure on the profile. The main new finding from this study is that chronic prenatal nicotine exposure, at doses which significantly reduced body weight, had no effect on the development of either anoxic gasping or bradycardia at any age. In addition we confirm that the main developmental characteristics of the response to anoxia can be summarized as follows: (a) the longest observed gasping duration was at 3 days. Duration and number of gasps declined in older rats; (b) anoxic gasping occurs at all ages up to 28 days; (c) the gasp rate is almost constant until death in the 3 day old rats but in older rats, gasping accelerates during the anoxic exposure; (d) regular QRS complexes continue long after anoxic gasping ceases; and (e) the heart rate falls rapidly at the onset of anoxia and then remains fairly stable until after gasping ceases. This stable, anoxic value for heart rate increased with age.
4.2. Significance of gasping Gasping is initiated and controlled by a mechanism that is anatomically and functionally distinct from the control of eupnea (St. John, 1990). Gasping and bradycardia are part of the autoresuscitation process, an important defense mechanism. (Gershan et al., 1990; Jacobi and Thach, 1989). Extreme hypoxia or anoxia rapidly induces apnea, which is then followed after a period varying from a few sec to a min by deep inspiratory gasps. Hypoxic hypometabolism probably also occurs (Mortola and Rezzonico, 1988; Mortola et al., 1989) Between gasps there is usually no movement, with low heart rate and little variability.
Gasps are associated with transient acceleration of heart rate. It is not clear whether the gasp-related acceleration is due to pulmonary stretch reflexes or is a direct effect of increased venous return. Chemoreceptor stimulation is not a factor in gasp-related cardiac acceleration, as acceleration occurs with gasps in an anoxic atmosphere. If a gasping rat is supplied with room air, the next gasp is soon followed by a rise in heart rate and a resumption of eupneic breathing (autoresuscitation) then occurs (Gershan et al., 1990). Gasping and bradycardia in response to anoxia/hypoxia appears to have two important benefits: apnea and bradycardia help in conserving oxygen, while the infrequent but intense inspiratory efforts can re-oxygenate the body if oxygen becomes available. Thus gasping forms part of a mechanism that allows infant mammals to survive periods of severe hypoxia. In infants, severe hypoxia may occur if for example the airway becomes obstructed by bedding and the infant fails to arouse. Gasping may then re-oxygenate the infant sufficiently for breathing to resume. Thus the ability to gasp, while normally unused, may become crucial for survival. Impaired ability to gasp would then reduce the infants chance of surviving a severe hypoxic challenge.
4.3. Rationale for methods In order to test our hypothesis, we induced gasping by anoxia. While complete anoxia is unlikely to occur naturally, the intention was not to model a real-life situation but simply to induce gasping rapidly and consistently. Apnea and gasping have occasionally been observed during severe hypoxia in our laboratory. Gasping is identical in hypoxia and anoxia, but analysis in hypoxia is complicated by the fact that the hypoxic animal can then re-oxygenate and resume eupneic breathing. For these reasons we used anoxia as a challenge. The study was not designed to measure survival time and no autoresuscitation was allowed. We have no direct evidence that the gasping animals would in fact have survived, as none of the animals was allowed to re-oxygenate. However, pub-
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lished data (Fazekas et al., 1941; Mott, 1961) and our own experience strongly suggest that survival is possible up to the last significant gasp and that the time to last gasp is therefore a measure of the survivable period of anoxia.
4.4. Confirmation of effecti6e dosing The main finding of this study was negative. No effect of nicotine on gasping occurred at any age. The question therefore arises whether the nicotine reached the fetuses in an effective dose. Mean maternal plasma nicotine during gestation was 134 ng/ml, compared with the range of 20 – 40 ng/ml found in human smokers (Lichtensteiger et al., 1988). Nicotine crosses the placenta readily and so there is every reason to believe the developing fetuses were exposed to nicotine: some studies have shown higher levels of nicotine in fetal than maternal plasma (Ruhle et al., 1995). Dose rates as low as 2 mg/kg per day have been shown to affect the development of cholinergic receptors in the CNS (Zahalka et al., 1992). Moreover, the nicotine-exposed rats were consistently lighter than control. This evidence strongly suggest that the fetal rats were exposed to sufficient nicotine to have significant physiological effects.
4.5. Factors influencing gasping time Our data show a marked decline in gasping time with age. This is consistent with previous published data that show survival times of 30 – 50 min in newborns, falling to less than 10 min by 2 weeks (Stafford and Weatherall, 1960; Mott, 1961). Neither the specific cause of gasping cessation, nor the decline in gasping time with age have been explained. The termination of gasping may be due to accumulation of neurotoxic amino acids. Valproate, which reduces the levels of aspartate and glutamate in the brains of mice, increases their survival time in anoxia (Thurston and Hauhart, 1989). However, most of the literature supports the idea that gasping ceases when energy substrates are exhausted, so that gasping stops earlier if metabolic rate is increased or if substrate stores are reduced. For example, Mott (1961) reported a linear relation between survival
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time and cardiac carbohydrate content in several species. Substrate exhaustion in respiratory muscle may also account for the postnatal decline in gasping time: a parallel postnatal decline in glycogen content of skeletal muscle and heart has been demonstrated (Shelley, 1961). More recently it has been shown that survival time of rats in severe hypoxia is directly related to their tissue glycogen content (Purshottam et al., 1978) and that diurnal variation in hypoxic survival is related to diurnal changes in the ability to mobilize liver glycogen (Kwarecki et al., 1984). Increasing the cerebral metabolic rate with aminophylline reduces gasp time in severed mouse heads (Thurston et al., 1978). Prolonged nicotine exposure has been shown to increase sympathetic nervous system activation of brown fat thermogenesis (Lupien and Bray, 1988). Although metabolic rate was not measured in these studies, thermogenesis is associated with increased metabolism, so that it might be expected that metabolic substrates would be exhausted earlier in nicotine-treated anoxic animals. Our finding that QRS complexes continued after the end of gasping agrees with previous studies (Stafford and Weatherall, 1960; Adolph, 1969). This observation appears to be inconsistent with the finding by Mott (1961) that survival time was related to energy substrates in the heart. However, QRS complexes may not be associated with significant contraction and stroke volume in conditions of extreme acidosis and tissue hypoxia, such as occur in our experiments, and we cannot be certain that cerebral perfusion persists in anoxia. Thus survival time may be limited by cardiac contractility, although electrical activity continues after gasping has ceased. Heart rate variability was not measured but appeared from a heart rate display to be very low during anoxia in both nicotine and control groups. If true, this would imply a low level of cardiac sympathetic activity under these conditions, as might be expected with an anoxic brainstem. Under these conditions the heart may receive essentially no neural input and be functionally denervated. However, we have no data on the neural control of the heart in anoxia. The specific system failure that causes cessation of gasping and heart beat in anoxia remains un-
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known, but we can safely conclude that it is unaffected by nicotine exposure.
5. Discrepancies with other literature There are some interesting discrepancies between our findings and some previous studies. All of our rats, at all ages, gasped in anoxia, with the youngest rats surviving for the longest time. This is in contrast to mice in which gasping ability showed a minimum at 17 – 23 days (Jacobi and Thach, 1989). Most other accounts of anoxic gasping, e.g. (Fazekas et al., 1941; Stafford and Weatherall, 1960) report that gasping time falls exponentially from newborn to adult and the reason for the anomalous responses of mice is unclear. No arrhythmias were observed in our studies. Bradycardia was established at the onset of anoxia and a low but steady heart rate persisted until after gasping ceased. As we did not attempt resuscitation we do not know if heart block would have occurred during re-oxygenation, but there was no evidence of heart block during gasping as has been reported (Gershan et al., 1992). Differences between strains of mice in their susceptibility to anoxic heart block have been reported (Gershan et al., 1992). Adolph (1969) noted that some ground squirrels restored breathing after anoxic apnea but failed to return to a normal heart rate and died: presumably these animals were in heart block, but no details are given. The apparent discrepancy between these findings and our study may have occurred because we did not attempt resuscitation. Gershan et al. (1992) reported that the probability of heart block in the less susceptible strain of mice increased with successive anoxic challenges, and Adolph (1969) observed it only during recovery. Thus, discrepancies between our studies and those of other workers are probably partly due to different protocols and partly indications of species differences. The physiological significance of such inter- and intra-specific differences, and their cause, is unclear. Adolph (1969) reported wide differences between species in both survival time in nitrogen and in the time course of its postnatal
change, with 50% survival for 10 min at ages ranging from 3 to 16 days. Tolerance to anoxia at birth was highest in the most immature newborns and the postnatal loss of tolerance occurred most rapidly in the fastest developing species. Clearly the loss of anoxia tolerance is part of a developmental process, but in rats it appears to be unaffected by nicotine exposure. In conclusion, prenatal exposure to nicotine, at doses that cause pre- and postnatal growth retardation, does not affect anoxic gasping or bradycardia in the rat pup at any age between 3 and 28 days. This finding is not consistent with our hypothesis that the increased risk of SIDS in infants of smoking mothers is mediated through an action of nicotine on the ability to survive and recover from extreme hypoxia. The mechanism by which maternal smoking increases the risk of SIDS remains unknown.
Acknowledgements Dr Schuen was supported by a Cystic Fibrosis Foundation training grant B856 (1994–1995) and an American Lung Association fellowship grant. Dr Carroll was supported by NIH HL02543-01. Dr Bamford was supported by a research grant from the SIDS Alliance (SP 0017).
References Adolph, E.F., 1969. Regulations during survival without oxygen in infant mammals. Respir. Physiol. 7, 356 – 368. Bamford, O.S., Schuen, J.N., Carroll, J.L., 1996. Effect of nicotine exposure on postnatal ventilatory responses to hypoxia and hypercapnia. Respir. Physiol. 106, 1 – 11. DiFranza, J.R., Lew, R.A., 1995. Effect of maternal cigarette smoking on pregnancy complications and sudden infant death syndrome. J. Fam. Pract. 40, 385 – 394. Eden, G.J., Hanson, M.A., 1987. Maturation of the respiratory response to acute hypoxia in the newborn rat. J. Physiol. (London) 392, 1 – 9. Fazekas, J.F., Alexander, F.A.D., Himwich, H.E., 1941. Tolerance of the newborn to anoxia. Am. J. Physiol. 134, 281 – 287. Gershan, W.M., Jacobi, M.S., Thach, B.T., 1990. Maturation of cardiorespiratory interactions in spontaneous recovery from hypoxic apnea (autoresuscitation). Pediatr. Res. 28, 87 – 93.
J.N. Schuen et al. / Respiration Physiology 109 (1997) 231–239 Gershan, W.M., Jacobi, M.S., Thach, B.T., 1992. Mechanisms underlying induced autoresuscitation failure in BALB/c and SWR mice. J. Appl. Physiol. 72, 677–685. Jacobi, M.S., Thach, B.T., 1989. Effect of maturation on spontaneous recovery from hypoxic apnea by gasping. J. Appl. Physiol. 66, 2384–2390. Janson, A.M., Hedlund, P.B., Hillefors, M., von-Euler, G., 1992. Chronic nicotine treatment decreases dopamine D2 agonist binding in the rat basal ganglia. Neuroreport 3, 1117 – 1120. Koos, B.J., Matsuda, K., Power, G.G., 1988. Fetal breathing and sleep state responses to graded carboxyhemoglobinemia in sheep. J. Appl. Physiol. 65, 2118–2123. Kwarecki, K., Debiec, H., Wroblewski, S., 1984. Biological time-related changes in tolerance of male rats to hypoxia– I. Survival rate and carbohydrate metabolism. Chronobiol. Int. 1, 239 – 244. Lichtensteiger, W., Ribary, U., Schlumpf, M., Odermatt, B., Widmer, H.R., 1988. Prenatal adverse effects of nicotine on the developing brain. Prog. Brain. Res. 73, 137–157. Lupien, J.R., Bray, G.A., 1988. Nicotine increases thermogenesis in brown adipose tissue in rats. Pharmacol. Biochem. Behav. 29, 33 – 37. Lynch, A.M., Bruce, N.W., 1989. Placental growth in rats exposed to carbon monoxide at selected stages of pregnancy. Biol. Neonate 56, 151–157. Mortola, J.P., Rezzonico, R., 1988. Metabolic and ventilatory rates in newborn kittens during acute hypoxia. Respir. Physiol. 73, 55 – 67. Mortola, J.P., Rezzonico, R., Lanthier, C., 1989. Ventilation and oxygen consumption during acute hypoxia in newborn mammals. A comparative analysis. Respir. Physiol. 78, 31– 43. Mott, J.C., 1961. Ability of young mammals to withstand total oxygen lack. Br. Med. Bull. 17, 144–148. Navarro, H.A., Seidler, F.J., Whitmore, W.L., Slotkin, T.A., 1988. Prenatal exposure to nicotine via maternal infusions. Effects on development of catecholamine systems. J. Pharmacol. Exp. Ther. 244, 940–944.
.
239
Purshottam, T., Kaveeshwar, U., Brahmachari, H.D., 1978. Hypoxia tolerance in rats in relation to tissue glycogen levels. Aviat. Space Environ. Med. 49, 1062 – 1064. Ruhle, W., Graf von Ballestrem, C.L., Pult, H.M., Gnirs, J., 1995. Correlation of cotinine level in amniotic fluid, umbilical artery blood and maternal blood. Geburtshilfe Frauenheilkd. 55, 156 – 159. Schoendorf, K.C., Kiely, J.L., 1992. Relationship of sudden infant death syndrome to maternal smoking during and after pregnancy. Pediatrics 90, 905 – 908. Shelley, H.J., 1961. Glycogen reserves and their changes at birth. Br. Med. Bull. 17, 137 – 143. Slotkin, T.A., Lappi, S.E., Tayyeb, M.I., Seidler, F.J., 1991. Chronic prenatal nicotine exposure sensitizes rat brain to acute postnatal nicotine challenge as assessed with ornithine decarboxylase. Life Sci. 49, 665 – 670. Stafford, A., Weatherall, J.A.C., 1960. The survival of young rats in nitrogen. J. Physiol. (London) 153, 457 – 472. St. John, W., 1990. Neurogenesis, control and functional significance of gasping. J. Appl. Physiol. 68, 1305 – 1315. Taylor, J.A., Sanderson, M., 1995. A reexamination of the risk factors for the sudden infant death syndrome. J. Pediatr. 126, 887 – 891. Thurston, J.H., Hauhard, R.E., Dirgo, J.A., 1978. Aminophylline increases cerebral metabolic rate and decreases anoxic survival in young mice. Science 201, 649 – 651. Thurston, J.H., Hauhart, R.E., 1989. Valproate doubles the anoxic survival time of normal developing mice. Possible relevance to valproate-induced decreases in cerebral levels of glutamate and aspartate and increases in taurine. Life Sci. 45, 59 – 62. Zahalka, E.A., Seidler, F.J., Lappi, S.E., McCook, E.C., Yanai, J., Slotkin, T.A., 1992. Deficits in development of central cholinergic pathways caused by fetal nicotine exposure. Differential effects on choline acetyltransferase activity and [3H]hemicholinium-3 binding. Neurotoxicol. Teratol. 14, 375 – 382.