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J Appl Physiol 102: 1429-1438, 2007. First published December 14, 2006; doi:10.1152/japplphysiol.00977.2006
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Postnatal maturation of laryngeal chemoreflexes in the preterm lamb

Marie St-Hilaire, Nathalie Samson, Elise Nsegbe, Charles Duvareille, François Moreau-Bussière, Philippe Micheau, Johann Lebon, and Jean-Paul Praud

Neonatal Respiratory Research Unit, Departments of Pediatrics and Physiology, Université de Sherbrooke, Sherbrooke, Quebec, Canada

Submitted 4 September 2006 ; accepted in final form 1 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Laryngeal chemoreflexes (LCR) are triggered by the contact of liquids with the laryngeal mucosa. In the mature organism, LCR trigger lower airway protective responses (coughing, effective swallowing, and arousal) to prevent aspiration. General belief holds that LCR are responsible for apnea and bradycardia in the newborn mammal, including humans. Our laboratory has recently shown that LCR in full-term lambs are consistently analogous to the mature LCR reported in adult mammals, without significant apneas and bradycardias (St-Hilaire M, Nsegbe E, Gagnon-Gervais K, Samson N, Moreau-Bussiere F, Fortier PH, and Praud J-P. J Appl Physiol 98: 2197–2203, 2005). The aim of the present study was to assess LCR in nonsedated, newborn preterm lambs born at 132 days of gestation (term = 147 days). The preterm lambs were instrumented for recording glottal adductor electromyogram, electroencephalogram, eye movements, heart rate, respiration, and oximetry. A chronic supraglottal catheter was used for injecting 0.5 ml of saline, distilled water, and HCl (pH 2) during quiet sleep, active sleep, and wakefulness on postnatal days 7 (D7) and 14 (D14). Laryngeal stimulation by water or HCl on D7 induced significant apneas, bradycardia, and desaturation, which, at times, appeared potentially life-threatening. No significant apneas, bradycardias, or desaturation were observed on D14. No consistent effects of sleep state could be shown in the present study. In conclusion, laryngeal stimulation by liquids triggers potentially dangerous LCR in preterm lambs on D7, but not on D14. It is proposed that maturation of the LCR between D7 and D14 is partly involved in the disappearance of apneas/bradycardias of prematurity with postnatal age.

sleep; control of breathing; apnea of prematurity; bradycardia

IN THE MATURE ORGANISM, STIMULATION of laryngeal mucosa receptors by liquids triggers highly protective reflexes [laryngeal chemoreflexes (LCR)], which consist of coughing, arousal, and effective swallowing to prevent subglottal aspiration. However, many animal experiments have led to the common belief that the LCR triggered in the newborn mammal are quite different and dominated by a vagal component, which includes laryngospasm, central or mixed/obstructive apnea, desaturation, and bradycardia (31). Consequently, LCR were believed to be partly responsible for apneas of prematurity, apparent life-threatening events, and probably some cases of sudden infant death syndrome (4, 20, 31, 32, 34).

Although many previous studies have assessed the LCR in newborn mammals, the majority of these have been performed in either anesthetized or sedated animals and/or were aimed at stimulating the subglottal area. In a recent study, we showed that stimulation of the laryngeal mucosa with water or an acid solution (HCl and citric acid, pH 2) did not induce any significant decrease in heart rate (HR) or respiratory rate (RR) in nonsedated, full-term lambs during quiet sleep (QS). However, significant lower airway protective responses (coughing, swallowing, and arousal) were observed following these stimulations, resembling the mature LCR reported in adult mammals (29). Accordingly, and in agreement with clinical observations that most healthy newborns do not present with apnea/bradycardia from laryngeal stimulation, we assumed that the development of life-threatening LCR is linked to some predisposing neonatal condition, such as preterm birth (3, 16, 22, 23). The aim of the present study was thus to test the hypothesis that potentially life-threatening cardiorespiratory events can be observed in preterm lambs during LCR, contrary to previous observations in term lambs. In addition, we tested the hypothesis that amplitude of those cardiorespiratory events decreases rapidly when preterm lambs reach a postconceptional age equivalent to full-term gestation. The LCR induced by various liquids (water, HCl, and saline) were comprehensively assessed throughout the various states of consciousness [QS, active sleep (AS), and wakefulness (W)] in nonsedated, preterm lambs at postnatal days 7 and 14. We believe that confirmation of these hypotheses, as demonstrated herein, brings forward important knowledge for the understanding of severe pathological conditions commonly observed in preterm infants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   Experiments were performed in six preterm lambs with a mean postconceptional age of 132 days (SD 1.3), range 131–134 days (normal gestation: 147 days), and a mean birth weight of 3.24 kg (SD 0.61) (range: 2.58–4 kg). The protocol of the study was approved by the Ethics Committee for Animal Care and Experimentation of our institution.

Preterm lamb model.   Six preterm lambs were delivered by cesarean section under epidural anesthesia with 5 ml of 2% lidocaine. The ewes received an intramuscular injection of betamethasone (0.5 mg/kg) 48 h before surgery. Immediately after birth, the lambs were intubated, and exogenous surfactant (10 ml of BLES, London, ON) was given by intratracheal injection (same dose repeated 24 h later). Intermittent positive pressure ventilation (12/6 cmH2O) was then administered through the endotracheal tube, using a Neopuff-Infant Resuscitator (Fisher-Paykel, Laguna Hill, CA) during 30 s or until initiation of spontaneous breathing. Standard care during the first 24 h of life systematically included continuous positive airway pressure through the endotracheal tube (6 cmH2O, Siemens Servo 300) for 24 h (first three preterm lambs) or 6 h (latter three lambs), with supplemental oxygen as needed to maintain transcutaneous oxygen saturation >95% [oxygen hemoglobin saturation measured by pulse oximetry (SpO2), Radical, Masimo, Irwine, CA]. In addition, the lambs were placed in an incubator for at least 24 h to maintain rectal temperature >38.5°C and received dextrose intravenous supplementation to maintain glycemia >2.3 mmol/l. Continuous nasogastric feeding with natural ewe's milk was initiated at 24 h of life and stopped for surgery (see below). Following surgery, all lambs received supplemental oxygen as needed to maintain SpO2 > 95%. Discontinuous nasogastric, then oral, feeding was initiated 24 h after surgery. Caffeine (one dose of 10 mg/kg subcutaneously) and/or doxapram (1 mg/kg intravenously, repeated when needed) was used as needed to treat severe, life-threatening apneas beginning 12–24 h after birth.

Instrumentation of the lambs and recording equipment.   Surgery was performed on the third day of life (3–4 days before the first recording) under general anesthesia (2% isoflurane, 30% N2O, and 68% O2). Atropine sulfate (0.1 mg/kg), ketamine (5 mg/kg), and ketoprofen (3 mg/kg) were injected intramuscularly before anesthesia, whereas 10 ml/kg lactate Ringer were infused intravenously throughout the surgical procedure. Following an intravenous loading dose of 25 µg/kg, morphine was infused (2 µg·kg–1·h–1) throughout surgery and for the next 18 h. Antibiotics (50 mg/kg ampicillin and 2.5 mg/kg gentamicin) were also injected intramuscularly before anesthesia and once a day throughout the study. Bipolar, gold-plated stainless steel electrodes were inserted into the thyroarytenoid (TA; a glottal adductor) through the lateral aspect of the thyroid cartilage for recording TA electrical activity [TA electromyogram (EMG)]. The electrode was then glued on the external surface of the thyroid cartilage. One bipolar electrode was usually placed in each TA muscle to ensure that at least one TA could be recorded in case of electrode damage. In three preterm lambs, a bipolar electrode was also inserted into the diaphragm for recording EMG, using an established technique (11). Two right-angled, platinum needle electrodes (E7–12, Grass Instruments, Quincy, MA) were inserted into the parietal cortex directly through the skull, at the level of the lambda suture, 1 cm from the midline, for electroencephalogram (EEG) recording. A platinum needle electrode (E2–12, Grass Instruments) was also inserted under the scalp as a ground. Two platinum needle electrodes (E2–12, Grass Instruments) were inserted subcutaneously on the proximal forelegs for recording electrocardiogram (ECG). Leads from all electrodes were subcutaneously tunneled to exit on the back of the lambs. Correct positioning of the electrodes in the TA and diaphragmatic muscles was systematically verified at autopsy. In addition, a supraglottal catheter was inserted transcutaneously to allow injection of liquids onto the larynx. The catheter was adapted from an infusion catheter and positioned such that its tip was 5–7.5 mm above the anterior part of the glottis. A plastic tubing (internal diameter 1 mm) was subcutaneously tunneled in the neck of the lamb and connected to the external part of the catheter, which protruded 15–20 mm at the level of the anterosuperior aspect of the thyroid cartilage (5). Correct catheter positioning above the glottis was monitored throughout the insertion procedure by direct laryngoscopy and was systematically confirmed during autopsy.

Lamb instrumentation was completed immediately before recordings in nonsedated lambs. Nasal airflow was recorded by use of a thermocouple wire (iron/constantan, type J; Omega Engineering, Stamford, CT) glued to the side of one nostril. Two platinum needle electrodes (E2–12, Grass Instruments) were placed subcutaneously into the outer upper region and the inner lower region of the right eye socket for electrooculogram (EOG) recording. Thoracic and abdominal elastic bands were placed for recording respiratory inductance plethysmography. An oximeter probe (8000R reflectance sensor, Nonin Medical, Plymouth, MN) was attached at the base of the tail for continuous monitoring of SpO2. Finally, the subcutaneous plastic tubing connected to the supraglottal catheter was attached to a custom-designed, radiotelemetry-driven injector, where the liquid to be injected was warmed and maintained at the lamb's body temperature.

Leads from the EMG, EOG, EEG, and ECG electrodes and the nasal thermocouple were connected to a transmitter attached to the lamb's back just before the experiment. Raw EMG, EOG, EEG, and ECG signals, as well as nasal flow, were transmitted by custom-designed radiotelemetry equipment (13). The raw EMG signals were moving time averaged (100 ms). Oxygenation was continuously monitored via a custom-designed pulse oximeter (SpO2) built from a Nonin OEM, with transmission by radiotelemetry (27). Thoracic and abdominal volume variations were qualitatively assessed using the Respitrace equipment (NIMS, Miami Beach, FL). All parameters were continuously recorded using acquisition software (Acknowledge version 3.2, Biopac System, Santa Barbara, CA). Collected data were stored on compact disk for further analysis.

Design of the study.   The study was designed to allow for simultaneous recording of TA and diaphragmatic EMG, ECG, EEG, EOG, nasal flow, sum of thoracic and abdominal movements, and SpO2, while triggering LCR by injection of liquids. Lambs were fed ad libitum with ewe's milk at 8:00 AM and 1:00, 6:00, and 10:00 PM and thrived normally. Lambs were comfortably positioned on a mattress without contention. Ambient temperature was maintained at 22°C and humidity at 70% throughout the experimental days. Recordings were performed in nonsedated lambs on postnatal day 7 or 8 (D7) and repeated on postnatal day 14 or 15 (D14). Each experimental day consisted of a random sequence of 0.5-ml injections via the supraglottal catheter of saline (0.9% NaCl, pH = 5.5, osmolarity = 326 mosM), distilled water (pH = 5.7), and hydrochloric acid (HCl) diluted in saline (pH = 2, osmolarity = 295 mosM). Choice of the solutions was dictated by the wish to compare results from the present experiment with those from our previous study in term lambs (29). Furthermore, the number of solutions studied was limited by the necessity to repeat injections in all three states of alertness, and by the design of both our protocol (random sequence) and the telemetered injector, which cannot be loaded with two solutions at the same time. All three solutions were injected once during W, QS, and AS (total of 9 injections on each experimental day). In addition, the catheter was systematically flushed with 1 ml of saline between each injection of test solutions (dead space of the catheter 0.5 ml). Each animal was given at least 15 min of recovery time between two injections. Events such as agitation, cough, and arousal or full awakening were noted by an observer throughout the recording sessions.

Data analysis.   The main objectives of the study were 1) to assess the characteristics of the LCR elicited by different solutions (HCl, water, and saline); 2) to assess the effect of the states of consciousness on the LCR; and 3) to assess the effect of postnatal maturation in nonsedated preterm lambs. In addition, as three lambs needed to be treated with caffeine for life-threatening apneas, results obtained in lambs treated with caffeine were compared with results in untreated lambs. Analysis of the LCR was performed as described previously (29). First, the cardiorespiratory responses to each laryngeal injection were assessed as follows. The percentage of decrease in HR [%dec HR = (HRBL – HRmin) * 100/HRBL] was calculated, with HRBL representing the baseline HR value averaged over 1 min before challenge, and HRmin representing the minimal HR value observed during the first minute after the challenge. Moreover, any presence of bradycardia was noted, as defined by a %dec HR >30%, as well as the number of bradycardias and total summed duration of bradycardias. The percentage of decrease in RR [%dec RR = (RRBL – RRmin) * 100/RRBL] was also calculated, with RRBL representing the baseline RR averaged 1 min before challenge, and RRmin representing the minimal RR value observed during the first minute after challenge. Time durations between the moment of injection and the moment of HRmin and RRmin occurrence (respectively HRmin and RRmin occurrence times) were measured for all stimulations. The presence of apneas (defined as at least two missed breaths relative to baseline breathing) was assessed within 1 min after laryngeal stimulation. The number of apneas and the total summed duration of apneas were noted. Moreover, the presence of any apneas longer than 5, 10, 20, and 30 s was also noted. Respiratory LCR duration was measured as the time duration between the onset of the LCR and resumption of three consecutive breaths (33). Moreover, any presence of desaturation was noted, as defined by a decrease in SpO2 of at least 4% or <90%. In addition to cardiorespiratory responses, the number of swallows (defined as a brisk, high-amplitude, and short-duration TA EMG burst) (25) occurring within the first minute after laryngeal stimulation was tallied. The time duration between stimulation and the first swallow, and the time duration between the first and last swallow (total swallowing duration) were measured. In addition, total summed duration of TA EMG (total TA EMG duration, indicating laryngeal closure) was calculated within 1 min after laryngeal stimulation for each stimulus. The presence of coughing was also noted (number of lambs which coughed/total number of lambs). Standard electrophysiological and behavioral criteria were used to define W, QS, and AS from EEG, EOG, and continuous visual observation (28). Cortical arousal from QS was defined by the association of a change in EEG (decrease in amplitude + increase in frequency) for 3 s or more, with at least two of the following modifications: a 10% increase in HR, or change in RR, or movement (10). Arousal from AS was recognized by direct observation of the lamb and disappearance of intense EOG activity. Full awakening was defined as when the lamb was still awake after 1 min (7). Finally, the number of stimulations with arousal or full awakening was documented.

Statistical analysis.   Measurements were averaged for the six preterm lambs as a whole. Values were then expressed as means (SD). Statistical analyses were performed using SAS (version 8.2, Cary, NC). The generalized estimating equations (GENMOD procedure) were used to estimate the effects of solution, state of alertness, and postnatal day on all parameters. Apart from proportion data (cough, arousal, and awakening), which followed a normal distribution, all of the response variables failed the Shapiro-Wilks test of normality and were then modeled as Poisson data. The working correlation structure chosen was of the exchangeable type to account for the correlation among the repeated measurements within lambs. A P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Baseline values obtained in each state of alertness on D7 and D14 are detailed in Table 1. A total of 54 laryngeal stimulations (6 saline, 6 distilled water, and 6 HCl in each state of alertness) were performed on D7, and 45 stimulations (5 saline, 5 distilled water, and 5 HCl in each state of alertness) on D14. Overall, mean weight increased from 3.9 (SD 0.3) on D7 to 4.5 kg (SD 0.3) on D14.


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  		Table 1. Baseline values for various stimuli during each state of alertness on postnatal days 7 and 14

 
LCR in QS on postnatal D7.   Results obtained during QS on D7 are detailed in Table 2. Following laryngeal stimulation, all solutions induced apneas and swallows within 3 s. Although not statistically significant, HRmin tended to be delayed compared with RRmin. Saline consistently induced apnea(s), swallowing and TA EMG activity, and some short bradycardias, but no coughing. Compared with saline, water elicited significantly greater cardiorespiratory responses and more swallows and TA EMG activity. Conversely, coughing was very rare, and arousal occurred in three of six lambs (P = 0.1 vs. saline). HCl elicited cardiorespiratory responses of intermediate amplitude between those of saline and water, including for %dec HR (P = 0.2 vs. saline and 0.6 vs. water), bradycardia duration (P = 0.9 and 0.04), %dec RR (P = 0.6 and 0.1), number of apneas (P = 0.9 and 0.1), and apnea duration (P = 0.2 and 0.8). Respiratory LCR duration following HCl injection was significantly longer than after saline and identical to that after water (P = 0.5). HCl induced significantly more swallows than saline and water. Total TA EMG duration was longer than after saline, but identical to that following water (P = 0.9). Weak coughing, occurring 20 s after HCl injection, was observed more often than after saline, but not after water (P = 0.3). Finally, while HCl induced arousal significantly more often than saline, the difference was not significant between HCl and water (P = 0.07).


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  		Table 2. Development of laryngeal chemoreflexes in response to various stimuli in quiet sleep on postnatal days 7 and 14

 
Detailed results on apnea and bradycardia duration observed in QS on D7 are shown in Table 3. Apneas longer than 20 s were observed only after water and HCl. Potentially life-threatening cardiorespiratory events were triggered in two preterm lambs following water and HCl, including apneas and bradycardia longer than 20 s and a decrease in SpO2 ranging from 13 to 36% (Fig. 1).


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  		Table 3. Apnea and bradycardia duration for each stimulus on postnatal days 7 and 14 in quiet sleep

 

Figure 1
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  		Fig. 1. A: potentially life-threatening cardiorespiratory reflexes triggered in preterm lambs at postnatal day 7, following instillation of 0.5 ml HCl onto the laryngeal mucosa during quiet sleep. The same pattern was observed following distilled water injection. From top to bottom: EEG, electroencephalogram; EOG, electrooculogram; Ta, electrical activity of the thyroarytenoid muscle; lung volume, sum signal of the respiratory inductance plethysmograph, allowing qualitative measurement of respiration (inspiration upward); ECG, electrocardiogram; HR, heart rate (beats/min). Note the prolonged, potentially life-threatening responses with repetitive apneas, bradycardia, and associated with desaturation down to 60%. B: laryngeal chemoreflexes in response to instillation of 0.5 ml HCl onto laryngeal mucosa at postnatal day 14 during quiet sleep (same lamb as in A).

 
LCR in QS on postnatal D14.   A clear maturation of the LCR was observed from D7 to D14 (Table 2 and Figs. 1 & 2). Following saline on D14, no bradycardias and almost no apneas were observed, and arousal tended to be more frequent (P = 0.1 vs. D7). Following water and HCl, cardiorespiratory events were less pronounced than on D7, and no potentially dangerous cardiorespiratory events were elicited (Table 3).


Figure 2
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  		Fig. 2. Effect of postnatal maturation on the percentage of decrease in respiratory rate (%dec RR; A), decrease in heart rate (%dec HR; B), and apnea duration (C) from postnatal day 7 (solid bars) to postnatal day 14 (shaded bars) in preterm lambs following instillation of 0.5 ml saline, distilled water, and HCl onto the laryngeal mucosa during quiet sleep. Values are given as means ± standard deviation. *P < 0.05.

 
Effects of sleep states on LCR on postnatal D7 and D14.   Results are presented in Tables 4 and 5. On D7, no major effects of sleep states were observed for any of the administered solutions, and the responses were very variable from one state to the other. While we were unable to pinpoint a specific pattern for each state of alertness, the following trends were observed. The cardiorespiratory responses to saline appeared identical in QS and AS, but weaker in W. On the contrary, the cardiorespiratory responses to water were greater in QS than in both W and AS. Finally, the cardiorespiratory responses to HCl were very variable, with a trend for being weaker in QS than in both W and AS. On D14, the results were also very variable, with the exception of apnea duration, which was longer in AS for all solutions.


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  		Table 4. Development of laryngeal chemoreflexes in response to various stimuli for each state of alertness on postnatal day 7

 

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  		Table 5. Development of laryngeal chemoreflexes in response to various stimuli for each state of alertness on postnatal day 14

 
Effects of prior caffeine treatment on LCR at postnatal D7.   Three out of six preterm lambs received caffeine treatment in the first 3 days of life for life-threatening apneas of prematurity. Although treatment was ceased at least 3 days before experimentation, plasma caffeine levels of 5, 7, and 7 mg/ml were still measured on D7. Interestingly, apnea duration and %dec HR appeared less pronounced following water and HCl in these lambs (Fig. 3), whatever the state of alertness. Arousal from QS or AS was not more frequent than in the lambs not treated with caffeine. Finally, no potentially dangerous cardiorespiratory reflexes were elicited in the three lambs treated with caffeine.


Figure 3
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  		Fig. 3. Effect of caffeine treatment on the %dec HR (A) and apnea duration (B) in preterm lambs at postnatal day 7, following instillation of 0.5 ml saline, distilled water, and HCl onto the laryngeal mucosa during quiet sleep. Solid bars, lambs with caffeine treatment; shaded bars, lambs with no caffeine treatment. Values are given as means ± SD. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The present study provides novel findings on LCR observed in preterm lambs following injection of various liquids, including the effect of early postnatal maturation. Overall, laryngeal stimulation by liquids in nonsedated, nontracheostomized preterm lambs on D7 induces clinically significant apneas, bradycardias, and desaturations, which can even be life-threatening in some cases. In addition, results show that a significant postnatal maturation of the LCR occurs in lambs born 2 wk prematurely, such that the LCR observed on D14 are much less prominent than those on D7. While the present study was not specifically designed to test this hypothesis, results suggest also that caffeine treatment blunts the cardiorespiratory components of the LCR. Finally, we were unable to show any clear effect of the states of alertness on liquid-induced LCR in the present study.

LCR in preterm lambs in QS at postnatal D7.   Extensive review of the literature reveals only one study on LCR in preterm newborn mammals (16). Distilled water injected through a tracheostomy in QS and AS at ~10 days of life in preterm lambs born at 130–135 days of gestation was shown to elicit apnea, bradycardia, and hypertension. The %dec RR and %dec HR observed in QS in that previous study, when no arousal occurred, were similar to our present observations on D7. Results of the present study thus confirm that exaggerated LCR can be elicited in preterm lambs, compared with term lambs (29). The present results obtained in lambs with intact airways, including life-threatening apneas on occasion, are in agreement with the prolonged cardiorespiratory responses previously reported in preterm, tracheostomized lambs in QS after water injection, including one lamb which failed to recover 45 s after stimulation and required resuscitation (16). The design of the present study does not allow us to infer the cause(s) of the exaggerated LCR observed in preterm lambs, compared with full-term lambs. Immaturity of the cardiorespiratory "controllers," increased laryngeal receptor sensitivity, and/or subglottal aspiration with stimulation of tracheal receptors may all be involved in preterm lambs to explain the differences with full-term lambs.

Aside from cardiorespiratory responses, laryngeal stimulation with liquids also elicits coughing, swallowing, and arousal. These components of the LCR are believed to be protective mechanisms aiming both at clearing liquids more rapidly from the laryngeal region and at preventing tracheal aspiration, which could lead to more dramatic cardiorespiratory events in the newborn. Such reflex responses were observed in full-term lambs during QS following water and HCl stimulation (29). In the present study, the few coughing responses observed in QS, especially following HCl, were different than those previously recorded immediately after liquid injection in full-term lambs: indeed, coughing was weaker, delayed (9–20 s after the stimulation), and occurred during central apnea in preterm lambs. Accordingly, coughing has been rarely observed after pharyngeal infusion of water (4% of the stimulations) in preterm human infants and generally occurred during a central apnea. Overall, coughing in preterm newborn mammals is not considered to be an adequate strategy for clearing liquids from the laryngeal mucosa (3, 31). Repeated swallows were also observed in preterm lambs in the present study, as well as in previously reported preterm human infants (3, 24, 25). In addition, swallows appeared more frequent than in full-term lambs (24, 29). An increase in bursts of nonnutritive swallowing occurring spontaneously has been reported previously in preterm infants and preterm lambs, regardless of the state of alertness (20, 24, 26). While the latter data have led to the speculation that preterm birth leads to an increase in pharyngo-laryngeal sensitivity, the physiological consequences of these repeated swallows still remain to be proven, with some authors considering them as ineffectual (24). Theoretically, arousal could play a critical role following laryngeal stimulation, especially by promoting more effective coughing and swallowing reflexes and cessation of apnea and bradycardia. While previous studies reported that arousal can be present in preterm lambs (16) and in preterm human infants (3), the present results suggest that arousal from QS tends to be less frequent in preterm lambs than in full-term lambs following saline or water injection (29). In addition, while some stimulations induced full awakening from QS, especially following HCl, none induced full awakening during AS, which may be less than ideal in conditions such as gastrolaryngeal reflux.

Results in QS on D7 in preterm lambs showing a tendency toward lower amplitude of the cardiorespiratory reflexes following HCl than water, together with a consistent tendency toward more coughing and arousal, suggest that the latter reflexes offer at least partial protective mechanism against HCl. In addition, the identical total duration of TA EMG, a marker of glottal closure, following water and HCl, suggests that the tendency toward greater cardiorespiratory reflexes with water vs. HCl does not originate from subglottal aspiration with stimulation of tracheal receptors. While the reasons for differences in the amplitude of the cardiorespiratory responses between water and HCl remain unknown, they are likely related to stimulation of different laryngeal receptors, as suggested by our recent observations of laryngeal receptors in newborn lambs known to be specifically stimulated by extracellular protons, including transient receptor potential vanilloid-1, acid-sensing ion channel-1, and twik-related acid-sensitive K+ channel-1 receptors (unpublished observations). However, it is noteworthy that, despite the fact that HCl tended on average to induce weaker cardiorespiratory responses than water, both HCl and water at times induced potentially life-threatening responses.

LCR induced by saline in preterm lambs.   Previous studies in newborn animals have shown minimal cardiorespiratory responses to the injection of saline on the laryngeal mucosa, and saline rinse has been used to inhibit responses to injection of liquids such as water, sodium bicarbonate, or acids (8, 15, 30). Accordingly, our laboratory has recently shown that saline induces virtually no responses in full-term lambs during QS (29). However, pharyngeal instillation of saline induced prolonged apneas in human preterm infants with apneas of prematurity (22, 23), although the apneas were more pronounced following water injection (3). In the present study, saline on D7 induced significant cardiorespiratory responses in preterm lambs, including clinically significant apneas and short bradycardias on a few occasions. The presence of cardiorespiratory responses to a bolus injection of saline onto the larynx may be related to stimulation of flow or pressure laryngeal mechanoreceptors (3, 30). Irrespective of the mechanism(s) involved, the present results confirm that premature birth is responsible for laryngeal hyperreflectivity to all studied liquids, including saline, and show that this hyperreflectivity includes both the apneic and the bradycardic component of the LCR.

Early postnatal maturation of the LCR in the preterm lamb.   It is generally accepted that LCR undergo a postnatal maturation from neonatal to adult life, with prolonged apneas, bradycardias, and bursts of swallows repeatedly reported in newborn mammals being replaced by coughing and arousal at adult age (see review in Ref. 31). However, studies of postnatal maturation of the LCR in the first weeks of life have yielded inconsistent results, which may be due to differences in species maturation or experimental conditions. Indeed, while severity of the apnea induced by laryngeal stimulation decreased during the first week of life in puppies in one study (2), another study reported that it first increased from the first to the third postnatal week in puppies and decreased thereafter (21). In addition, other studies have shown that the apneic responses following laryngeal stimulation are minimal in piglets older than 50 days, compared with piglets younger than 15 days (6, 12). Results of the present study further show that the exaggerated cardiorespiratory responses observed in preterm lambs on D7 are significantly decreased on D14, with no life-threatening responses observed at that later age. Of note, the necessary use of caffeine in three out of the six lambs at D7 may have even obscured the amplitude of this maturation effect. Overall, the reflexes observed on D14 were similar to the reflexes observed in term lambs on D4 (29). Not surprisingly, the two lambs, which had potentially life-threatening reflexes on D7 in response to water and HCl, still had the most important reflexes on D14. These results are in agreement and extend previous studies performed in preterm and full-term human infants, which showed much more prolonged responses with repetitive swallows, apnea, and laryngeal closure in preterm (3, 23, 24) than in full-term infants (24).

Effects of caffeine treatment on the LCR.   Xanthine derivatives such as caffeine or theophylline are widely used for treating apneas of prematurity (1). The effects of xanthines, which are potent adenosine-receptor blocking agents, include an increase in minute ventilation and in CO2 sensitivity and a decrease in hypoxic depression and periodic breathing (18). Apneas induced by injection of milk or water onto the larynx were shown to be shortened following administration of aminophylline in piglets (12). As alluded to previously, although the present study was not designed to adequately test this hypothesis, our results suggest that caffeine blunts the cardiorespiratory components of the LCR induced by saline, water, and HCl in preterm lambs. If confirmed, these observations would be especially relevant to the management of apneas of prematurity.

LCR and states of alertness.   The present study did not reveal any effect of the states of alertness. While some statistical analyses yielded significant differences, the results were inconsistent from one stimulus to the other, and/or from D7 to D14. One previous study in preterm lambs found longer bradycardia and apnea duration in both AS and QS than in W, when no arousal was induced (16). Our inability to confirm these previous results may be related to the low statistical power in the present study. This is due to the complexity of our preterm ovine model, which precluded studying many lambs, and the technical constraints of our telemetered injector, which did not allow for testing each stimulus repeatedly in every state of alertness. Clearly, the absence of any effect of the state of alertness on the LCR components must not be considered as a definitive result.

Clinical implications.   Apneas of prematurity are usually defined for clinical purposes by a cessation of breathing accompanied with bradycardia and/or hemoglobin oxygen desaturation (17). Both clinical experience and results from a few studies in preterm human infants strongly suggest that LCR are one of the mechanisms involved in apneas of prematurity (see review in Ref. 32). In addition to feeding and the administration of oral medications such as polyvitamins or ranitidine, endogenous stimuli, such as gastrolaryngeal refluxes and upper airway secretions, can trigger LCR in preterm infants (19, 32). As to the relevance of acid-triggered LCR, the preterm infant has been shown to have the capacity to maintain gastric pH < 4 (9), and a recent study has confirmed that, between feedings, 25% of gastric refluxes are acidic in healthy preterm infants (14). The present study clearly demonstrates that the contact of an acid solution at pH 2 with the laryngeal mucosa (surrogate for an acid gastrolaryngeal reflux) can trigger life-threatening reflexes in some nonsedated preterm lambs in the first week of age, which is clearly at variance with our previous observations for full-term lambs. In addition, the observation in preterm lambs of significant responses to saline, a stimulus similar to airway secretions (23), suggests that upper airway secretions/saliva can be responsible for LCR with significant cardiorespiratory responses in preterm newborn infants. However, given that most gastric refluxes in the newborn infant are postprandial and nonacidic, the present results must be furthered by studies of LCR triggered by milk. Finally, we propose that postnatal maturation of the LCR, as shown from D7 to D14 in preterm lambs, constitutes one of the mechanisms involved in the postnatal disappearance of apneas of prematurity when reaching a postconceptional age corresponding to full-term gestation.

In conclusion, our results show that preterm birth amplifies the cardiorespiratory events observed during LCR and favors the development of life-threatening LCR in lambs. Such laryngeal hyperreactivity significantly decreases when lambs reach a postconceptional age equivalent to full-term gestation. The present results strongly suggest that LCR is involved in the apneic/bradycardic events observed in premature human infants.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
M. St. Hilaire is a scholar of the Canadian Institutes for Health Research. J.-P. Praud is a national scholar of the Fonds de la Recherche en Santé du Québec. The study was supported by grants from the Canadian Institutes for Health Research (NRF 15558) and the Quebec Foundation for Research into Children's Diseases.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors gratefully acknowledge the expert technical assistance of Christophe Grenier and Jean-Philippe Gagné, and the assistance of Marie-Pierre Garant (MSc, Biostatistics) for statistical analyses. BLES Inc., London, ON, graciously provided the surfactant.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-P. Praud, Dept.s of Pediatrics and Physiology, Université de Sherbrooke, J1H 5N4 QC, Canada (e-mail: Jean-Paul.Praud{at}USherbrooke.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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