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Departments of 1 Physiology, 3 Medicine, and 2 Pediatrics, Dartmouth Medical School, Lebanon, New Hampshire 03756
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ABSTRACT |
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We tested the hypothesis that inhibition of neurons within the rostral ventral medulla (RVM) would prolong the laryngeal chemoreflex (LCR), a putative stimulus in the sudden infant death syndrome (SIDS). We studied the LCR in 19 piglets, age 3-16 days, by injecting 0.05 ml of saline or water into the larynx during wakefulness, non-rapid eye movement (NREM) sleep, and REM sleep, before and after 1 or 10 mM muscimol dialysis in the RVM. Muscimol prolonged the LCR (P < 0.05), and the prolongation was greater when the LCR was stimulated with water compared with saline (P < 0.02). The LCR was longer during NREM sleep than during wakefulness and longest during REM sleep (REM compared with wakefulness). Muscimol had no effect on the likelihood of arousal from sleep after LCR stimulation. We conclude that the RVM provides a tonic facilitatory drive to ventilation that limits the duration of the LCR, and loss of this drive may contribute to the SIDS when combined with stimuli that inhibit respiration.
sudden infant death syndrome; sleep
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INTRODUCTION |
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A TRIPLE-RISK MODEL HAS BEEN proposed for the sudden infant death syndrome (SIDS) in which a sequence of events leading to death is triggered during sleep when a vulnerable infant is exposed to an exogenous stressor during a critical period of development (12). Among many possible exogenous stressors identified in epidemiological and physiological studies (17, 19, 42), we have been interested in the laryngeal chemoreflex (LCR). The LCR is elicited when fluid stimulates laryngeal mucosal receptors. The reflex response may involve swallowing, coughing, apnea, bradycardia, hypertension, redistribution of blood flow, and arousal from sleep (15, 16, 20). The manifestations of the LCR seem to evolve over the course of development: swallowing and apnea are prominent in preterm infants, swallowing remains in full-term neonates, but the duration of apnea declines during this period, and cough may emerge as a more prominent element of the response in adult animals (47). Arousal from sleep is common, but not universal, when the reflex is elicited; arousals tend to be less frequent during active sleep (36, 43). The respiratory disruption and apnea associated with the LCR have led to speculation that the LCR may contribute to the pathogenesis of the SIDS (10, 17, 41). Furthermore, prone positioning of infants reduced swallowing and enhanced the respiratory disruption associated with the LCR during active sleep (19).
A number of neurotransmitter receptor deficits have been identified in certain nuclei in the brain stems of infants who died of the SIDS (22, 38, 39). To understand the function of the neuroanatomic abnormalities identified in babies dying of SIDS, we turned to studies of neonatal piglets. We focused on the rostral ventral medulla (RVM) in these animals. We defined the RVM as a region within 1.2 mm of the ventral surface that extends rostrocaudally the length of the facial nucleus and mediolaterally ~1.5-4.0 mm from the midline. This area includes the retrotrapezoid nucleus, the parapyramidal region, and the juxtafacial parts of the nucleus paragigantocellularis lateralis. Thus many of the nuclei in which decreased neurotransmitter receptor binding was found in babies dying of the SIDS are included in the RVM, and the RVM in piglets may be homologous with parts of the arcuate nucleus in humans. Inhibition of neurons within the RVM after dialysis of 10 mM muscimol, a GABAA agonist, reduced the ventilatory response to 5% inhaled CO2 in neonatal piglets during wakefulness and non-rapid eye movement (NREM) sleep (5) and disrupted the architecture of sleep in these animals (8). In decerebrate piglets, muscimol dialysis in the RVM enhanced the respiratory inhibition associated with activation of baroreceptors (6), and cooling of the ventral medullary surface enhanced respiratory inhibition after superior laryngeal nerve stimulation in anesthetized piglets (26). The foregoing studies suggest that functional deficits in the RVM may reduce respiratory stability in that a response to a ventilatory stimulant (CO2) was blunted, but a response to ventilatory inhibition [elevated blood pressure (BP)] was enhanced. In the present study, we tested the hypothesis that muscimol dialysis in the RVM would enhance the inhibitory action of the LCR on respiration in neonatal piglets. We elicited the LCR by instilling either 0.9% saline or water into the larynx during wakefulness, NREM sleep, and rapid eye movement (REM) sleep before and after muscimol dialysis in the RVM.
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METHODS |
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Experiments were performed on 28 piglets ranging in age from 3 to 16 days (8.3 ± 0.7 days; means ± SE) with an average weight of 2.4 ± 0.1 kg (means ± SE). The Institutional Animal Care and Use Committee of Dartmouth College approved all surgery and experimental protocols.
Surgical instrumentation. Anesthesia was induced with isoflurane in O2, and surgery was performed under sterile conditions. A femoral venous catheter was inserted, and each piglet was treated with intravenous antibiotics (20 mg/kg iv, cefazolin). A dual-lumen umbilical catheter (4-Fr, 40 cm; Vygon, Ecguon, France) was inserted into the femoral artery and advanced until the tip was in the abdominal aorta. A thermistor was placed subcutaneously lateral to the abdominal midline. Two wire electromyographic (EMG) electrodes were sewn into the diaphragm through a subcostal incision in the right upper quadrant of the abdomen to measure diaphragmatic EMG (EMGdia) activity. A second set of EMG wires was inserted into the genioglossus through a submental incision to measure genioglossal EMG (EMGgg) activity. A 2.7-mm-diameter catheter was placed in the trachea just below the cricoid cartilage (feeding tube, 8-Fr, 16 in., Kendall, Mansfield, MA) to record endotracheal pressure. This catheter was also used for tracheal fluid injections in a subset of animals. The wires and catheters were tunneled subcutaneously and exited the skin at the top of the skull.
At this point in the surgery, each animal was rotated and placed in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA). Stereotaxic coordinates were taken for the lambda, bregma, and a specific point on the right ear bar. These coordinates were used to place a microdialysis probe in the RVM (44). The microdialysis guide tube and stylet (Bioanalytical Systems, West Lafayette, IN) were inserted through a burr hole in the skull so that the tip of the guide tube was just dorsal to the RVM. The 250-µm-diameter microdialysis probe tip, which was 1 mm longer than the microdialysis guide tube, protruded into the RVM. EEG electrodes were placed over the left frontal, right occipital, and right parietal regions. The frontal and occipital electrodes were used to measure EEG activity, and the right parietal electrode was used as a grounding electrode. Electrooculographic (EOG) electrodes were placed lateral to and just above each eye. A pair of EMG wires was placed in the neck muscles posteriorly. All wires exited on the top of the head and were attached to brass fittings and placed in two plastic pedestals. The pedestals and the microdialysis guide tube were cemented to the skull (Cranioplastic Powder, Plastics One, Roanoke, VA). To stimulate the LCR, we placed a pharyngeal catheter through the nose at the time of each experiment. At the time of surgery, we sutured a nose ring, made from the proximal 0.5 cm of the hub of a hypodermic needle, into one nostril. At the end of the surgery, a polyethylene tube was passed through the nose ring into the larynx. The larynx was visualized with a laryngoscope, and we noted the length of the nasal catheter at which the tip of the nasal catheter lay just caudal to the epiglottis but rostral to the esophagus and the aryepiglottic folds. At the conclusion of surgery, 0.1 mg/kg buprenorphine was administered to provide postsurgical analgesia. After surgery and at the end of each experimental day, each piglet was returned to the sow in the animal care facility. Every day after surgery, piglets were treated with oral antibiotics mixed into their formula (250 mg levofloxacin). The surgical incisions were treated daily with a topical antibiotic ointment (Bacitracin).Measurements. The animals were first studied ~24 h after surgery. Respiration was measured by using a barometric plethysmograph modified to allow continuous gas flow (40). A reference chamber with a slow leak was connected to the plethysmograph to minimize pressure fluctuations associated with changes in room pressure (2). Air flowing through the plethysmograph was heated (~38°C) and fully humidified. The average box temperature was between 25 and 26°C. Respiratory activity was derived from the pressure fluctuations in the box, which were measured with a model DP103 transducer (Validyne, Northridge, CA). All catheters and recording wires were passed through a sealed port in the side of the plethysmograph.
One lumen of the arterial catheter was connected to a transducer to measure arterial BP (model BP-1, WPI, Sarasota, FL). The second lumen was used to withdraw blood-gas samples without disrupting the BP record. EEG and EOG signals were amplified and band-pass filtered (0.1-300 Hz). The neck EMG, EMGgg, and EMGdia were amplified and band-pass filtered from 10 to 300 Hz. The fractional O2 content of inlet and outlet air (model S-3A/II, Applied Electrochemistry, Pittsburg, PA) and fractional content of CO2 in the outlet air (Capstar-100, CWI, Ardmore, PA) were determined. Plethysmograph and animal temperatures were continuously measured (YSI, Yellow Springs, OH). All signals were digitized at 1,000 Hz and recorded by using a computerized data-acquisition system (PowerLab, ADInstruments). Throughout the experiment, a video image of each piglet was recorded on videotape.Protocol. Animals were studied for 1-4 days after surgery. One and one-half hours before the experiment, the plethysmograph was sealed to allow the temperature and humidity to stabilize. The plethysmograph was calibrated by sequential triplicate injections of 1, 2, 3, and 5 ml of air. During the calibrations, the piglet was brought into the laboratory setting and fed to increase the likelihood of sleep during the experiment. The piglet was placed prone in a sling in the box and connected to the monitoring equipment. The stylet of the microdialysis guide tube was removed and replaced with a microdialysis probe. Artificial cerebrospinal fluid (aCSF) was passed through the dialysis probe at 8.5 µl/min (this dialysate flow rate caused no volume transudation of fluid across the dialysis membrane). Before each experiment, one side of the piglet's nose was anesthetized with 1 ml of 2% lidocaine jelly. A double-lumen nasal catheter (4-Fr double-lumen PICC, Braun Medical) was passed into the larynx to the appropriate depth determined during surgery, and the catheter was tightly attached to the nose ring. Once the nasal catheter was in place, the piglet was placed in the box and acclimated to the box for ~1 h.
After this stabilization period, the LCR was studied. A computer-controlled syringe pump (KdScientific, New Hope, PA) was used to inject either 0.05 ml of water or 0.05 ml of 0.9% saline at 7 ml/min into the larynx through one or the other side of the dual-lumen nasal tube. One lumen was used exclusively for water and the other exclusively for saline, and the sequence of saline and water stimuli was randomized. The dead space of the catheter was substantial compared with the volume of injections. Because much of the catheter lay within the airway of each piglet, the fluids were warmed to the temperature of the pharynx before injection. In preliminary experiments, we tried injection volumes as large as 0.2 ml, but behavioral responses, a startle reaction followed by significant movement of the animal, precluded adequate repetitive testing of the LCR in multiple sleep states. Injection volumes of 0.05 ml were the smallest volume that consistently produced a LCR and behavioral responses that were either absent or mild. Injections were timed to coincide with the end of inspiration, and we waited a minimum of 3 min between injections. During the experiment, the sleep state of each piglet was determined on the basis the behavior of the animal, the EEG and EOG signals, neck EMG, and respiratory and BP recordings (described below). We tried to distribute the injections equally across wakefulness and NREM and REM sleep, and we tried to give at least two injections of saline and water in each sleep state before and after muscimol. We studied the LCR in an initial control period during which aCSF alone was dialyzed into the RVM. It took 1-2 h to obtain control data, and once sufficient injections were made, 1 or 10 mM muscimol were added to the dialysate. The muscimol was dialyzed for 30 min, after which the dialysis was changed back to aCSF. We began studying the effect of 10 mM muscimol (4 piglets), but this dose of muscimol disrupted sleep (8). Also, we were particularly interested in LCR responses during sleep, so we reduced the dose of muscimol in the dialysate to 1 mM (15 piglets). The effect of muscimol on the LCR was studied for 1-2 h, starting at the conclusion of the muscimol dialysis. The effect of muscimol is long lasting, and we, therefore, always studied the effect of muscimol after a control period of dialysis. To assess the effect of time alone, we conducted control experiments in which aCSF was dialyzed continuously, but the LCR was studied at the same times as in the muscimol studies.Data analysis: definition of the LCR. We measured the duration of the LCR as our primary index of the strength of the reflex. The duration of the LCR was determined from the respiratory tracings of the plethysmograph EMG, EMGgg, and EMGdia activity and fluctuations in endotracheal pressure. The onset of the LCR was taken from the onset of inspiratory activity of the breath during which the saline or water injection was made. The mixture of events that constitute the LCR may include swallowing, apnea, coughing, a startle, and arousal from sleep and movement of the entire head, and most of these events disrupt the regularity of the respiratory pattern. We defined the end of the LCR as the time at which a regular pattern of breathing was restored, and the onset of a regular respiratory pattern was defined by the occurrence of five uninterrupted breaths. This definition of LCR duration encompasses both apnea and irregular and obstructed breathing caused by swallows or coughs, but these events were not explicitly included in our definition of the LCR, except to the extent that these events prevented the restoration of regular breathing. Apneas were defined as the cessation of respiratory activity for a time greater than the duration of the two breaths that preceded the breath during which fluid was injected into the larynx.
Cardiorespiratory and sleep state scoring variables.
Breath-to-breath tidal volume, respiratory frequency, and minute
ventilation (
E) were calculated from
plethysmographic pressure fluctuations. EEG signals were resampled at
100 Hz and filtered with a bandwidth of 0.1-30 Hz. The EEG record
was divided into 5-s epochs, and absolute spectral power density was
computed over a frequency range of 0-50 Hz, as described
previously (8). The power for each epoch was averaged over
the delta (0.1-4.0 Hz), theta (5.0-9.0 Hz), or sigma
(10.0-14.0 Hz) frequency bands. Periods of wakefulness, NREM
sleep, and REM sleep were defined by comparing continuous plots of
tidal volume, mean arterial pressure, heart rate, delta, theta, and
sigma EEG power density, EOG movements, and eye openings. Wakefulness
was defined by the presence of low-amplitude fast EEG activity, low
delta power, and the presence of nuchal EMG activity. The piglets also
had intermittent eye opening and gross body movements during
wakefulness. NREM sleep was defined by periods of high delta power and
high EEG amplitude. During NREM sleep, the piglet often showed signs of
shivering, which completely disappeared during REM sleep. REM sleep
periods were characterized by the presence of REM, nose and ear
twitching, an irregular pattern of breathing, a drop of mean arterial
BP, low delta power, a high-frequency EEG signal, and loss of nuchal EMG activity. Arousals were defined by marked sleep state transitions (NREM to wakefulness and REM to wakefulness) lasting
10 s. We did not
define microarousals lasting <10 s because we saw them so
infrequently; when arousal was part of the response to fluid injection,
the arousal tended to be long lasting.
Neuroanatomy.
At the conclusion of experiments, each piglet was killed with an
injection of 1.5 ml/kg pentobarbital sodium followed by 5-10 ml of
saturated potassium chloride administered intravenously. Microinjections of 20-50 µl of 1% potassium permanganate were made into the RVM through a broken microdialysis probe passed through
the microdialysis guide tube to mark the location of the tip of the
microdialysis probe and site of dialysis (the distribution of the
permanganate was not used in any way to judge the anatomic distribution
of muscimol dialysis). The permanganate formed an insoluble tar, which
was not removed during subsequent tissue processing (44).
The ventral surface of the brain was photographed to provide a
permanent record of the site of muscimol microdialysis with respect to
external brain stem landmarks. The external landmarks were correlated
with the location in computer reconstructions and further analyses of
cut sections. The brain stem was removed from the animal, placed in
cryoembedding medium (Tissue-Tek OCT 458, Sakura Finetek, Torrance,
CA), and frozen in isopentane at
70°C. Brain stems were sectioned
(50 µm) in a cryostat at
18°C, and sections were mounted on
gelatinized glass slides. Sections were fixed over night in 10%
formalin in phosphate-buffered saline (pH 7.0) and stained with cresyl
violet (1, 28).
Analysis and statistics.
We studied 19 piglets during muscimol dialysis. We tried to study each
piglet on multiple days, and we succeeded in studying four piglets on 3 days, seven piglets on 2 days, and the remainder on 1 day only. We
stimulated the LCR >1,500 times in these animals. We treated each
study day as a separate observation, but the responses of each piglet
within each day were averaged within each treatment variable (sleep
state and type of fluid injected before and after muscimol treatment).
Thus we had 34 study days or observations on 19 piglets (we obtained
identical statistical results when we averaged multiple days from each
piglet and treated each piglet as an observation). The individual
average values that made up an observation day were entered into a
three-way repeated-measures ANOVA (Systat 9.0, Evanston, IL) in which
the type of fluid (water or saline), the muscimol dose (0, 1, or 10 mM), and sleep state (wakefulness, NREM sleep, or REM sleep) were
within-subject factors. When the ANOVA indicated that significant
differences existed among treatment conditions, we performed multiple
preplanned comparisons using the Bonferroni adjustment of P
values. A similar statistical approach was used to analyze control
studies in which the LCR was tested at the same times as in the
muscimol studies, but no muscimol was added to the aCSF dialysate. We
analyzed the effect of muscimol, the type of fluid used to stimulate
the LCR, and sleep state (REM or NREM sleep) on the likelihood of
arousal from sleep after stimulation of the LCR using a series of
2 tests. We also compared the effect of
injection of fluid above and below the larynx in three piglets. We used
an ANOVA in which route of injection, volume injected, and the presence
or absence of muscimol in the dialysate were between-subject variables.
Finally, we tried to correlate the location of the dialysis within the brain stem with the magnitude of the change in LCR duration after muscimol dialysis. We did this using cluster analysis (K-means procedure, Systat 9.0) in which the percent change in the LCR was
associated with particular sets of rostrocaudal, dorsoventral, and
mediolateral coordinates that described the location of the dialysis
probe within the brain stem (6). Values are expressed as
means ± SE, and the criterion for statistical significance was
set at P
0.05.
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RESULTS |
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Characterization of the LCR.
An example of the LCR, which demonstrates the multiple facets of the
reflex response, is shown in Fig. 1.
Swallowing and coughing occurred frequently, and apneas, when they
occurred, often followed a period of swallowing or coughing rather than
being at the onset of the LCR. We did not see consistent bradycardia
during the apneas after laryngeal stimulation of the LCR. Although not
shown well in Fig. 1, the LCR often had a stuttering appearance in
which a few normal-appearing respiratory efforts occurred before
further swallowing, coughing, or apnea occurred. For this reason, we
required five regular breaths to define the termination of the LCR. The example also demonstrates the typical pattern of EEG arousal. We did
not see coughing without prior arousal, just as others have reported
previously (43).
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Effect of muscimol on LCR duration during wakefulness and sleep. The effects of 1 mM muscimol dialysis and 10 mM muscimol dialysis on the LCR duration were not different, and the results from these two muscimol doses were combined. We were unable to obtain data during REM sleep during control or test conditions on 12 observation days, and the repeated-measures ANOVA is intolerant of missing data. Therefore, we excluded REM sleep in our initial analysis and compared wakefulness and NREM sleep. We had complete data for this analysis from 30 study days. Muscimol prolonged the LCR (P < 0.05), and the prolongation was significantly greater during NREM sleep than during wakefulness (P < 0.05). The LCR was longer when elicited by water compared with saline (P < 0.001), and this effect was also significantly greater during NREM sleep compared with wakefulness (P < 0.001). We studied piglets ranging in age from 3 to 16 days, and we included age as a covariate in the ANOVA. We found no evidence that the age of the piglet modified the response of the LCR to muscimol, to the type of fluid injected, or to the sleep state. Thus inhibition of small regions within the RVM prolonged the LCR during NREM sleep, and water was a more effective stimulus than saline.
We tried to explore the effect of REM sleep on the LCR by analyzing the LCR elicited by saline and water separately. We had complete data from all states for 21 study days after saline injection and 22 days after water injection. The results of these statistical comparisons are presented in Fig. 2. The LCR after saline injection was similar in all conditions: the particular sleep state and the presence or absence of muscimol treatment did not seem to modify the response to saline. On the other hand, the duration of the LCR elicited by water was significantly prolonged after muscimol when all sleep states where considered together, and the LCR increased progressively as the piglets moved from wakefulness to NREM sleep to REM sleep. The LCR during REM sleep was significantly prolonged compared with wakefulness (P < 0.01), although, in this analysis of water responses only, the LCR prolongation comparing wakefulness with NREM sleep was not statistically significant. Thus the LCR elicited by water was prolonged during sleep, particularly during REM sleep; muscimol prolonged the LCR elicited by water injection into the larynx; but the response to saline was not affected by sleep state or muscimol treatment.
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E in the period
before the LCR was stimulated, both before and after muscimol dialysis.
There were no changes in the pattern of breathing within sleep states,
and only the
E is presented in Table
2 as a function of sleep states and
presence or absence of muscimol in the dialysate. There was no effect
of muscimol treatment on resting ventilation. However, there were
significant sleep state effects on
E.
E was greatest in wakefulness, reduced slightly, but
not significantly, during NREM sleep, and substantially reduced during
REM sleep compared with both NREM sleep (P < 0.001)
and wakefulness (P < 0.001). Thus there was a
reduction in
E among sleep states that paralleled the sleep state-related prolongation of the LCR after instillation of
water in the larynx.
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Anatomic locations of dialysis probes.
The anatomic locations of the tips of the dialysis probes used to
administer muscimol are shown in Fig. 3
(triangles, 1 mM muscimol; circles, 10 mM muscimol dialysis; whether
the symbols are open or solid reflects the magnitude of the LCR, see
below). On the left side of Fig. 3, the location of each
dialysis probe was projected onto the ventral surface of a piglet's
brain stem. The grid reflects the approximate position of the facial
nucleus. On the right side of Fig. 3, probe locations were
placed on hemisections of the medulla at the appropriate mediolateral,
dorsoventral, and rostrocaudal location. In previous studies using 10 mM fluorescein to mimic the distribution of muscimol, the region
affected by dialysis for 10-20 min was ellipsoidal and had a
volume of ~5.8-6.3 µl (4, 33). The volume of
distribution of 1 mM muscimol would be substantially smaller. We had
hoped that, in some probe locations, the LCR would be prolonged, but we
expected that some of the probe locations would fall outside the RVM
and not affect the LCR duration. However, most of the probe locations
were within the RVM. Nonetheless, some of the piglets demonstrated much
greater prolongation of the LCR than others. To determine whether the
location within the RVM correlated with the magnitude of LCR
prolongation, we calculated the average percent change in the LCR for
each piglet across all sleep states (wake and NREM and REM sleep) after
stimulation with distilled water. In 15 piglets, the LCR was prolonged,
and the average prolongation was 53 ± 6% (means ± SE;
range 13-88%; open symbols), and, in four piglets, the LCR was
shortened by 10 ± 5% (range 1-22%; solid symbols). We
performed a cluster analysis to determine whether the magnitude of the
LCR response was correlated with the location of the dialysis probe,
and we found no evidence that any region within the volume of the brain
stem that we studied was either especially effective or ineffective in
modifying the LCR duration after muscimol dialysis.
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LCR response during time control studies.
Muscimol is a long-acting drug, and we added muscimol consistently as
the second treatment in the foregoing experiments. To make sure that
the passage of time alone was not responsible for the prolongation of
the LCR that we found after muscimol treatment, we conducted control
studies in nine piglets. The timing of these control studies was
identical to the muscimol studies, but, at the time muscimol would have
been added to the dialysate, we simply continued the dialysis with
aCSF. A three-way ANOVA identical to the analysis of test data was
used, and the results of this analysis are shown in Fig.
4. There was no effect of time in these studies. As in the previous set of studies, water was a more effective stimulus than saline (P = 0.05), and there was a
significant main effect of sleep (P < 0.001). The LCR
was longer during REM sleep than NREM or wakefulness, but none of these
specific comparisons among sleep states was significant once the
P values were adjusted for multiple comparisons. Thus there
was no evidence that time alone altered the LCR response over the
duration of our experiments. The locations of the dialysis probes used
in these control experiments are also shown in Fig. 3 (diamonds and X
marks). The probe locations were, in general, more caudal than some of
the probes used in the muscimol studies, but the control locations
were still within the RVM.
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Arousal responses before and after muscimol dialysis.
Arousal forms an important part of the LCR and may have some survival
advantage. Therefore, we used a series of
2 tests to
assess the effect of type of fluid injected, sleep state (NREM vs. REM
sleep; data from wakefulness were excluded), and the presence or
absence of muscimol dialysis on the likelihood of arousal after
stimulation of the LCR. Because dialysis of 10 mM muscimol into the RVM
disrupts normal sleep architecture and reduces the occurrence of REM
sleep (8), we performed this arousal analysis only on data
obtained before or after dialysis with 1 mM muscimol. We performed a
series of stratified multiway comparisons using the Mantel-Haenszel
test, but only the main effects of the treatments that we studied
revealed significant differences. As a result, the simpler
2 tests on the pooled data from all of the arousals
elicited are presented in Table 3. Not
surprisingly, given the apparent greater effectiveness of water
compared with saline as a stimulus for the LCR, arousal was
significantly more likely after injecting water into the larynx than
saline (Table 3; P < 0.001). Arousal was more likely
from NREM sleep than REM sleep (Table 3; P < 0.05).
However, 1 mM muscimol had no effect on the likelihood of arousal
(Table 3).
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Response to sublaryngeal stimulation of the LCR.
Apnea is a common feature of the LCR in studies of anesthetized animals
(23, 25), but uncommon in unanesthetized sleeping piglets
(36). The increase in the occurrence and duration of apnea
may be attributed in part to anesthesia (37) but the route of administration also differs among these studies. To test the hypothesis that a sublaryngeal route of administration of fluid might
enhance the likelihood or duration of apneas, we performed further
studies in three of the piglets used in the time control studies. We
administered saline or distilled water, either through the nasal
catheter or through the catheter used to measure tracheal pressure,
which was 2-3 cm below the larynx. An example of the response to
sublaryngeal administration of 0.15 ml of distilled water during NREM
sleep in the control period before muscimol was administered is shown
in Fig. 5. Note the immediate onset of
apnea and bradycardia late in the first apnea. The respiratory disruption was prolonged, and repetitive apneas were punctuated by
coughing, swallowing, and brief bursts of respiratory effort.
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0.1 ml, and a small volume as <0.1 ml. The
results of this study are shown in Fig.
6, and the location of the dialysis
probes in these three animals is shown in Fig. 3 (X marks). Injections
administered below the larynx (trachea) prolonged the LCR more
effectively than injections delivered into the larynx
(P < 0.001). Large-volume injections significantly prolonged the LCR when given into the trachea before muscimol dialysis
(P < 0.05), but, after muscimol was given, the LCR was significantly longer only after small-volume tracheal injections. The
route of administration was a far more important factor determining the
duration of the LCR than the volume of fluid injected. Furthermore, almost all of these injections resulted in arousal, so we detected no
difference in arousal frequency based on the site of injection.
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DISCUSSION |
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We tested the hypothesis that inhibition of small regions of the RVM after muscimol dialysis would prolong the LCR. In general, the results confirmed this hypothesis. In addition, we confirmed previous studies in which water was a more effective stimulus of the LCR than saline, and the potency of water as a stimulus was greater in NREM sleep than in wakefulness and greater still in REM sleep. Apnea was a more prominent feature of the LCR after tracheal injection than after laryngeal injection. The modulatory effect of inhibition of neurons within the RVM is a novel finding that is probably relevant to the pathogenesis of the SIDS.
Stratification of the LCR response. The manifestations of the LCR vary as a function of animal age (47) and appear to vary in neonates, depending on the type and location of the stimulus and on the sleep state of the newborn. Some aspects of the LCR are conservative, in the original sense of the word. Apnea, bradycardia, and redistribution of blood flow conserve limited oxygen reserves without removing or reversing the inciting stimulus. Other aspects of the LCR seem more concerned with removing the offending stimulus and restoring airway patency and function. Swallowing and coughing, for example, clear the airway, and arousal may be necessary to permit coughing (43). However, the conservative and restorative aspects of the reflex are, to some extent, mutually exclusive; apnea precludes coughing, for example. Therefore, the respiratory control system seems to perform a triage in which restorative or conservative aspects of the reflex function sequentially, or one aspect is emphasized more than the other. The choice of restorative vs. conservative responses seems to vary as a function of the strength of the stimulus. We found that saline was relatively ineffective as a stimulus. Swallowing and coughing were elicited, but the respiratory disruption was mild, and apnea was less common after saline stimulation. Water in the larynx was more potent, but most potent was water injected below the larynx. We saw significant apnea and bradycardia after tracheal instillation, although saline was still less effective than water. This pattern suggests to us that restorative aspects of the LCR are the first line of defense, but, to the extent that they are ineffective, conservative aspects of the reflex become more prominent. The graded response of the LCR may arise from the persistence of the stimulus as restorative mechanisms fail and conservative responses develop. This stratification of responses is consistent with the observation that apneas often followed swallowing when injections were made in the larynx. The early onset of prolonged apneas after tracheal stimulation indicates that conservative responses need not follow failed restorative mechanisms. The immediate onset of apneas raises the possibility of two classes of receptors in which laryngeal stimulation elicits the restorative elements of the LCR, but tracheal stimulation fosters the occurrence of apnea. To the extent that restorative airway clearance mechanisms fail, laryngeal stimulation might lead to tracheal stimulation as liquid leaks from the larynx into the trachea. The failure of even large volumes of water injected above the larynx to elicit significant apnea suggests that a large area of stimulation alone may not initiate apnea and favors the hypothesis that tracheal receptors may have some specificity for the apneic response. Finally, we saw no consistent bradycardia accompanying the apneas after laryngeal stimulation. Bradycardia was apparent only during apneas and only when the apnea duration was prolonged after tracheal stimulation of the LCR. A similar lack of consistent bradycardia was reported in normoxic infants and preterm infants who, nonetheless, developed significant apnea after water was infused into the hypopharynx (41, 51). The bradycardia may not be part of the LCR, but it may develop during longer apneas as a result of hypoxia and carotid body stimulation (51).
Effect of sleep state on the LCR and arousals.
Sleep modified the duration of the LCR. The LCR was generally longest
during REM sleep, especially when water was used to stimulate the LCR.
The LCR was of intermediate duration during NREM sleep and shortest
during wakefulness.
E was also least in REM sleep,
intermediate in NREM sleep, and greatest during wakefulness. Thus some
of the prolongation of the LCR may have been due to the sleep
state-dependent reduction in ventilatory drive. In general, there is an
inverse relationship between the duration of the LCR and respiratory
drive in unanesthetized animals. For example, hyperoxia prolongs the
LCR in awake and sleeping piglets (49). Others have also
noted the decline in respiratory drive associated with different sleep
states and the greater severity of the LCR as respiratory drive
diminishes in newborn lambs (29). Stimulation of muscle
afferents, which stimulate ventilation, also shortens the LCR in lambs
(14). The effect of hypoxia on the LCR is less clear cut.
Hypoxia may prolong the LCR in anesthetized piglets and exacerbates the
apnea and bradycardia associated with the reflex (23), but
other studies, also in piglets, suggest that hypoxia shortened apnea
duration (52). Hypoxia shortens the apneic response to
electrical stimulation of the superior laryngeal nerve in decerebrate
piglets (7), but it prolongs the apnea associated with
laryngeal infusion of water in infants (51). Thus, whereas
it is appealingly simple to suggest that the duration of the LCR is
inversely related to the level of respiratory drive (and some data,
particularly during sleep, support this concept), the reality is more
complicated, especially when one considerers the effects of hypoxia.
Modulation of the LCR by dialysis of muscimol in the RVM. Dialysis of muscimol prolonged the LCR when water was used to stimulate the reflex, and the prolongation was greatest in REM sleep. These findings raise two issues: what is the specific mechanism whereby muscimol modifies the LCR, and, in the larger scheme of the structure and organization of the brain stem, what is the function of the RVM with respect to cardiovascular and respiratory reflex responses? The LCR could be prolonged because the clearance mechanisms are less effective. Muscimol might modify the muscular function of swallowing or coughing and reduce the efficiency of these processes. Our EMG and tracheal pressure recordings are too crude to address this issue; such an analysis would require something like the radiological assessment used by others (31). Furthermore, muscimol seemed to delay the onset of the clearance mechanisms. The onset of swallowing, apnea, coughing, and arousal were all delayed after muscimol in our subsidiary analysis of the elements of the LCR (Table 1). The reflex seemed to occur in slow motion. Either of these mechanisms, delayed onset or reduced clearance efficiency, might prolong the reflex by slowing clearance of the substances stimulating the LCR. Furthermore, tract tracing studies demonstrated that connections exist between the RVM and the motoneurons of the laryngeal musculature that might plausibly permit an interaction between the RVM and swallowing mechanisms (50). On the other hand, the progressive prolongation of the reflex from wakefulness to NREM sleep to REM sleep might reflect the state-related reduction in the respiratory drive to breathe. The sleep state-related changes in ventilation are also correlated with sleep state-related changes in CO2 chemosensitivity (5): the ventilatory response to CO2 is least during REM sleep, intermediate in NREM sleep, and slightly greater during wakefulness. Dialysis of 1 mM muscimol reduced the ventilatory response to CO2 in a pattern identical to the effect of 10 mM muscimol (L. van der Velde, J. Roberts, A. K. Curran, R. A. Darnall, D. Bartlett, Jr., and J. C. Leiter, unpublished observations). Furthermore, the duration of the LCR was reduced in anesthetized humans (34) and anesthetized piglets (24) during exposure to hypercapnia, and hypercapnia blunted the inhibition of ventilation associated with superior laryngeal nerve stimulation in anesthetized piglets (26). Thus the mechanism of action of muscimol to prolong the LCR may be correlated with reduced sensitivity to CO2. Finally, reduced efficiency of LCR clearance mechanisms and reduced ventilatory drive are not mutually exclusive, and both of these mechanisms might contribute to prolongation of the LCR.
Before concluding that inhibition of central CO2 chemosensory mechanisms contributes to LCR prolongation, it is worth considering the effect of inhibition of the RVM on other cardiovascular and respiratory reflexes. In our laboratory's previous studies, 10 mM muscimol reduced the ventilatory response to CO2 during wakefulness and NREM sleep when dialyzed into the RVM (5). However, muscimol dialyzed into the RVM also inhibited the ventilatory response to 8 or 10% hypoxia in unanesthetized piglets (18) and enhanced the inhibition of respiration after stimulation of baroreceptors with phenylephrine in decerebrate piglets (6). Thus inhibition of neurons within the RVM need not be specific to CO2 chemosensory mechanisms. The alternative hypothesis is that muscimol may act by CO2-independent mechanisms. Forster and colleagues used cooling thermodes placed bilaterally on the ventrolateral surface of the medulla to inhibit neural activity in awake and anesthetized goats (13, 35). Inhibition of neural activity by cooling the ventral surface of the brain stem was equally effective during hypoxia, hypercapnia, and exercise; the inhibitory effect of cooling was not specific to the hypercapnic ventilatory response. Increasing the depth of neural inhibition into the ventral medulla by prolonging the duration of cooling produced more selective inhibition of the hypercapnic ventilatory response. Similar results were obtained in neonatal goats (27). In this scheme, muscimol may have reduced the ventilatory response to CO2, enhanced baroreceptor-mediated respiratory inhibition, and prolonged the LCR by reducing a tonic, CO2-independent excitatory drive to breathe that also varies as animals move from wakefulness to NREM sleep to REM sleep.Variation of the LCR among and within piglets. Among the 19 animals receiving muscimol, the LCR was prolonged in 15 "responders," but the LCR was slightly shorter in four piglets. One of the nonresponders received 10 µM muscimol, and three received 1 µM muscimol. The lack of a consistent dose response in either the responders or nonresponders was a little surprising, but it suggests to us that the proximity of the dialysis probe with respect to the neurons is more important than the drug dose, given the relatively small volume of distribution of the drug. This interpretation is also consistent with the cluster analysis of anatomic locations that we performed. Nonresponders and responders did not differ with respect to neuroanatomic coordinates. Thus we believe that neurons that modulate the LCR (and other reflexes) are not distributed homogenously but that they exist in small clusters spread heterogeneously throughout the RVM (6).
We studied 11 piglets on multiple days. The LCR among the nonresponders (n = 2) was consistently unaffected by muscimol treatment. Among the responders, two piglets had no response on 1 day, but the average LCR duration over all treatment days was still prolonged. In the remaining seven piglets, the LCR was consistently prolonged on all study days. We replaced the nasal cannula each day of the experiment, and it is possible that the location of the nasal cannula, and, therefore, the effectiveness of the stimulus, varied day to day in those piglets studied on more than 1 day.Implications for SIDS. The triple-risk model for the pathogenesis of SIDS states that three factors contribute to the death of each infant: an underlying vulnerability, a critical period of homeostatic control, and an exogenous stressor (12). We studied the LCR, an exogenous stressor that inhibits ventilation, in neonatal piglets with an experimentally induced vulnerability (inhibition of the RVM by muscimol dialysis). The results of the study are consistent with the triple-risk model of SIDS in that inhibition of the RVM prolonged the LCR and enhanced the disruption of a stable respiratory pattern. The LCR seems particularly relevant to SIDS (19, 45, 46). Esophageal reflux may stimulate the reflex, and the LCR may be less effective in the prone position (swallowing was reduced and respiratory disruption was enhanced when the LCR was elicited during active sleep in the prone sleeping position; Ref. 19), which may tie the epidemiology of sleeping position to this particular exogenous stressor. Furthermore, the results of the present study provide further evidence that neurons within the RVM sustain and stabilize respiratory activity, and to the extent that the neurons are absent or deficient in some function, the ventilatory responses to inhibitory stimuli will be enhanced, and responses to excitatory stimuli will be blunted.
GABA receptors are ubiquitous, and muscimol dialysis may mimic, to some extent, the loss of neurons observed in the arcuate nucleus in some babies who died of SIDS (11, 30). However, the neurotransmitter receptor defects identified in the SIDS are specific to muscarinic, kainite, and serotonergic receptor binding within multiple regions of the brain stem (22, 38, 39), and the effect of muscimol is not specific for any of these neurotransmitter defects. Given the effect of muscimol on ventilatory responses to hypercapnia, hypoxia, baroreceptor stimulation, and the LCR, we are presently conducting studies of the specific neurotransmitters implicated in studies of infants dying of SIDS. In summary, we demonstrated that muscimol dialysis in an extended region of the RVM prolongs the respiratory disruption associated with the LCR in waking and sleeping neonatal piglets. We believe that a facilitatory input to respiratory drive, which may or may not be related to CO2 sensitivity, originates or is integrated within the RVM and promotes respiratory stability. Loss of this tonic excitatory drive to breathe prolongs the LCR, reduces the ventilatory response to CO2, and enhances the respiratory inhibition associated with baroreceptor stimulation. These findings support the triple-risk model of the SIDS and suggest that the neurotransmitter defects described in babies who died of the SIDS may be associated with impairment of physiological mechanisms that stabilize respiratory output.| |
ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical assistance of Laurie Hildebrandt, Hong Gao, and Dr. Man-Hua Sun.
This work was supported by National Institute of Child Health and Human Development Grant HD-36379, the American Heart Association, and the Charles H. Hood Foundation. A. K. Curran is a Parker B. Francis Fellow in Pulmonary Research.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. C. Leiter, Dept. of Physiology, Dartmouth Medical School, Lebanon, NH 03756 (E-mail: james.c.leiter{at}dartmouth.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/japplphysiol.01103.2002
Received 2 December 2002; accepted in final form 3 January 2003.
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