| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Pediatrics, Respiratory Research Group, Faculty of Medicine, The University of Calgary, Calgary, Alberta, Canada T2N 4N1
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Recently, Wong et al. (Wong KA, Bano A, Rigaux A, Wang B, Bharadwaj B, Schurch S, Green F, Remmers JE, and Hasan SU, J Appl Physiol 85: 849-859, 1998) demonstrated that fetal lambs that have undergone vagal denervation prenatally do not establish adequate alveolar ventilation shortly after birth. In their study, however, vagal denervation was performed prenatally and the deleterious effects of vagal denervation on breathing patterns and gas exchange could have resulted from the prenatal actions of the neurotomy. To quantify the relative roles of pre- vs. postnatal vagal denervation on control of breathing, we studied 14 newborn lambs; 6 were sham operated, and 8 were vagally denervated below the origin of the recurrent laryngeal nerve. Postoperatively, all denervated animals became hypoxemic and seven of eight succumbed to respiratory failure. In vagally denervated lambs, expiratory time increased, whereas respiratory rate, minute ventilation, and lung compliance decreased compared with the sham-operated animals. In the early postoperative period, the frequency of augmented breaths was lower but gradually increased over time in the denervated vs. sham-operated group. The dynamic functional residual capacity was significantly higher than the passive functional residual capacity among the sham-operated group compared with the denervated group. No significant differences were observed in the prevalence of various sleep states and in the amount of total phospholipids or large- and small-aggregate surfactants between the two groups. We provide new evidence indicating that intrauterine actions of denervation are not required to explain the effects of vagal denervation on postnatal survival. Our data suggest that vagal input is critical in the maintenance of normal breathing patterns, end-expiratory lung volume, and gas exchange during the early neonatal period.
augmented breaths; fetus; functional residual capacity; gas exchange; hypoxemia; pulmonary mechanics; pulmonary surfactant; sighs; sleep states
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
AS OPPOSED TO EPISODIC FETAL breathing movements, breathing becomes continuous at birth. The mechanisms of the establishment of continuous breathing remain unknown. Evidence suggests that vagal innervation plays an important role in the maintenance of normal postnatal breathing patterns and alveolar ventilation (7, 12-14, 50). In these studies, however, vagal denervation was performed in the cervical region, which could have resulted in the compromise of upper airway function, including vocal cord paralysis. Furthermore, the animals in these studies were either anesthetized, tracheotomized, and/or studied after the immediate newborn period (7, 14, 32, 48, 50).
To avoid the limitations associated with the previous studies, Wong et al. (57) performed intrathoracic vagal denervation prenatally to investigate the role of vagal innervation on the establishment of breathing and gas exchange at birth. In this study, sham-operated animals established effective gas exchange, whereas vagally denervated animals developed profound respiratory failure. However, prenatal vagal denervation might have had confounding effects on a number of physiological adaptations occurring during transition from fetal to neonatal life, including increases in pulmonary blood flow, surfactant secretion, and lung liquid absorption (18, 26).
Although the study by Wong et al. (57) provided unequivocal evidence that vagal innervation is critical for the establishment of continuous breathing and adequate gas exchange at birth, the mechanisms for the postnatal respiratory failure remain unclear. Two possibilities exist: 1) the intrauterine vagal denervation may have impaired fetal lung development (1), or 2) after birth, vagal denervation may have compromised breathing, most likely by eliminating the afferent feedback from the lung. Because Wong et al. performed vagal denervation 10-14 days before birth, both types of effects could have contributed. On the other hand, evidence suggests that vagal innervation plays an important role in the regulation of breathing during the early postnatal period. Consequently, elucidating the contribution of the vagal afferents in establishing and maintaining breathing and pulmonary gas exchange immediately after birth represents an area of research of fundamental significance to neonatal respiratory control.
To exclude the confounding effects of prenatal vagal denervation on breathing patterns and gas exchange, we performed bilateral intrathoracic vagal denervation during the early postnatal period. We hypothesized that vagal innervation is critical for pulmonary gas exchange and maintenance of normal breathing patterns in the early neonatal period.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Surgical procedures. All procedures were performed in accordance with the Canadian Council on Animal Care, and the study protocol was approved by the animal care committee of the University of Calgary. Fourteen newborn lambs underwent either intrathoracic vagal denervation (n = 8) or sham surgery (n = 6) within 24 h of birth. Surgery was performed under general anesthesia using 4% halothane in oxygen for induction and 1.5% halothane for maintenance. With the use of sterile techniques, a 2-cm incision lateral to the trachea and immediately below the thyroid cartilage was made in the neck to expose the jugular vein and carotid artery. Polyvinyl catheters (1 mm ID, 2 mm OD; Portex, Hythe, Kent, UK) were inserted 7 cm into the jugular vein and carotid artery and secured in place. The arterial catheter was used to draw blood samples for arterial pH and blood-gas tension measurements and to record arterial blood pressure and heart rate, whereas the venous catheter was used to administer antibiotics and fluids intra- and postoperatively. During surgery, rectal temperature (39°C) and arterial pH (7.35-7.45) and blood-gas tensions [arterial PCO2 (PaCO2) = 35-45 Torr and arterial PO2 (PaO2) = 90-110 Torr] were maintained by adjusting the heating pad temperature and ventilator settings, respectively.
After implantation of vascular catheters, the lamb was placed on its left side and a 2-cm incision was made at the fourth intercostal space. The vagus and phrenic nerves were identified, and a 2-cm section of the right vagus nerve was removed below the origin of the recurrent laryngeal nerve. To minimize accumulation of intrapleural air, an 8-Fr chest tube (Argyle) was inserted through a small incision made at the sixth intercostal space, and a purse-string suture was placed around the incision site. The procedure was then repeated on the left side. Through a 2-cm incision made parallel to the ribs at the 10th intercostal space, three diaphragmatic electrodes were implanted into the right costal diaphragm. Although three diaphragmatic electrodes are implanted, only two electrodes (bipolar) are used to obtain the optimum diaphragmatic signal. All the incisions were closed in layers using size 0 silk. Finally, both chest tubes were attached via a "Y" connector and placed under water seal at
12 cmH2O.
After vagal denervation was performed, lambs were placed in prone
position to implant electrodes to record electrocorticogram (ECoG),
electrooculogram (EOG), and nuchal electromyogram (EMGNK). The surgical details for electrode placement have been given elsewhere (24). Briefly, the two ECoG electrodes were placed on the
dural surface, 2 cm caudal to the coronal suture line and 3 cm apart through two holes drilled in the skull. To record EMGNK, a
3-cm incision was made in the dorsal neck region, and pair of
electrodes were sewn into one of the right nuchal muscles. Two 0.5-cm
incisions were made along the superior and inferior orbital ridges and
EOG electrodes were implanted as described previously
(23). ECoG, EOG, and EMGNK were used to define
various sleep states as described previously (23). All
electrode wires (AS 633 Cooner, Chatsworth, CA) were subcutaneously
tunneled, secured, and soldered to a Lemo connector (Lemo, Ecublens,
Switzerland) for diaphragmatic and sleep-state recordings.
If the animals were unable to establish effective pulmonary gas
exchange and spontaneous breathing after surgery, supplemental oxygen
and/or manual positive pressure ventilation were administered. Effective pulmonary gas exchange was defined as arterial pH >7.30, PaCO2 <50 Torr, and PaO2 >50 Torr
(41). Establishment of spontaneous breathing was defined
as a respiratory rate of at least 15 breaths/min.
Augmented breaths were measured using a catheter filled with saline
placed in the midesophagus. In the current study, two definitions of
augmented breaths were used: 1) a biphasic spontaneous inspiration having at least a twofold increase in esophageal pressure (30, 53) and 2) a biphasic response with at
least 50% increase in esophageal pressure and diaphragmatic EMG
(EMGdia) compared with the preceding 10 breaths. We used
both definitions, since a twofold increase in tidal volume has been
used previously but on a purely arbitrary basis. In contrast, an
increased and biphasic nature of esophageal pressure reflects
respiratory center output and is more relevant to our current study
especially in view of the changes in pulmonary compliance. The
esophageal pressure was continuously recorded in both sham-operated and
vagally denervated animals.
The animals were considered awake and alert when they were able to open
their eyes and lift their heads. To help maintain normal body
temperature (39.0°C), we used a neonatal transport incubator during
transfer from the operating room to the laboratory and a heat lamp
during observations in the laboratory.
Experimental design.
The experimental design is given in Fig.
1. Postoperation sleep states,
EMGdia, arterial blood pressure, heart rate, esophageal pressure, and rectal temperature levels were continuously recorded. The
ECoG, EOG, EMGNK, and EMGdia signals were
amplified and filtered appropriately with frequency ranges of
0.5-10 Hz, 5-40 Hz, 50 Hz to 1 kHz, and 50 Hz to 1 kHz,
respectively. Arterial blood pressure and heart rates were recorded
using a pressure transducer (Statham P23 ID; Gould Instrument Division,
Cleveland, OH). Arterial blood samples were drawn every 60-120 min
or more frequently if clinically indicated for measurement of arterial
pH, and blood-gas tensions were corrected for body temperature (IL 1312 blood-gas manager). No arterial blood samples were withdrawn while the
lambs were receiving supplemental oxygen. Esophageal pressure was
obtained by placing an 8-Fr feeding tube in the midesophagus and then
recorded using a pressure transducer (Statham P23 ID; Gould). All
bioelectric signals were displayed on an eight-channel chart recorder
(Gould Brush 2800s), digitized, and stored on a videocassette using an eight-channel Neurocorder (DR-886; Neurodata Instruments, New York,
NY).
|
1 · day1 to prevent
hypoglycemia and dehydration. Two doses of 25 mg/kg of cefazolin sodium
in saline (Ancef, Smith Kline Beecham Pharma, Oakville, ON) and 2.5 mg/kg of gentamicin sulfate (Garamycin injectable, Schering Canada,
Pointe-Claire, PQ) were administered intravenously every 8 h.
Pulmonary function tests were performed using a 4.5- or 5-Fr
endotracheal tube 1 h before surgery, during recovery, and 6 and
24 h after surgery or earlier if respiratory failure
occurred. The animals were only intubated during the pulmonary
function tests, each lasting ~10-15 min. The rationale for
intubation was to avoid air leak and to maintain the accuracy of the
data during lung compliance and resistance measurements. The animals
were extubated immediately after each pulmonary function test was
completed. With the exception of pulmonary function tests, all data
were collected while the animals were breathing spontaneously with intact upper airway. Neither assisted ventilation nor continuous positive airway pressure was used once the animals were extubated. The
recovery period was defined as the time when the animal was breathing
spontaneously. A Fleisch pneumotachograph (size 00), a Hans-Rudolph
flow occluder (Hans Rudolph, Kansas City, MO), and Validyne pressure
transducers (DP45-32-A-3-5-S-4-D and DP45-14-A-3-5-S-4-D, Validyne
Engineering, Northridge, CA) were used to measure tidal volume,
respiratory rate, minute ventilation, inspiratory and expiratory times,
and static and dynamic compliance and resistance. Data were stored on a
PC (Dell 233 MHz) and then analyzed with the use of the Anadat, Labdat,
and Auto programs (version 5.2, RHT-Info Dat, Montreal, PQ).
For calculation of compliance, resistance, and the time constant, the
pressure signal during occlusion and the flow signal during exhalation
were used. The data were recorded for 4 s with a sampling rate of
250 samples per second for each channel. The high sampling rate was
necessary to record the rapid changes in the flow signal in small
volumes, especially in noncompliant lungs. Static respiratory system
compliance and resistance were measured by occluding airflow at end
inspiration from 10 breaths in each subject for each period of
pulmonary function testing (29). Dynamic lung compliance
and pulmonary resistance were calculated using the computerized method
of multiple linear regression (29). To obtain information
regarding lung volume and expiratory braking, flow-volume graphs were
constructed after end-inspiratory occlusions for 300 ms. After
occlusion, the flow signal was integrated to obtain volume and a
flow-volume curve was constructed. The slope of the curve was
established by using regression analysis with a regression coefficient
greater than 0.99. The regression line (slope) was then extrapolated to
obtain the volume that represents the complete emptying of the lung to
the normal functional residual capacity (FRC), whereas the
y-intercept (flow) of the regression line provides the peak
flow (see Fig. 6). The static lung compliance was calculated as the
relation of occlusion pressure to the extrapolated volume and the total
lung resistance as the peak flow to the occlusion pressure.
The animals were euthanized using Euthanyl (MTC Pharmaceuticals,
Cambridge, ON) either 24 h postoperatively or earlier if severe
respiratory failure occurred (pH < 7.0). At autopsy,
sectioning of both vagi and the integrity of the phrenic nerves were
confirmed in all animals. The right middle lobe was tied using size 4 silk to avoid leakage of bronchoalveolar lavage as described below. A
1-cm3 section of the right middle lobe was removed and
fixed with 1% osmium tetroxide in fluorocarbon and postfixed in 2.5%
freshly prepared gluteraldehyde. The lung tissues were embedded in
epon, sectioned and stained with uranyl acetate/lead citrate, and
examined using a Hitachi 7000 transmission electron microscope for
presence or absence of type II cells and lamellar bodies in a blinded fashion.
Bronchoalveolar lavage was performed on both lungs except the right
middle lobe while still intact. Saline (100 ml/kg) was infused using
the gravitational method at 20 cmH2O, intratracheally in
four aliquots as described previously, and gently withdrawn using a
60-ml syringe. The gravitational method was used to minimize precipitous lung distension to avoid lung rupture and an inadvertent surfactant release (43). The lavagate was centrifuged for
8 min at 150 g to remove cells and debris. The supernatant
was then centrifuged for 20 min at 40,000 g (Ti 60 rotor,
Beckman) to separate the lavagate into large and aggregates of
surfactant. Large aggregates were resuspended in 15 ml of saline and
analyzed for total phospholipids using Bartlett's method
(3). Surface tension-lowering properties were assessed
using the captive bubble technique as described by Schürch et al.
(49).
After the lavage, the lungs were fixed using formalin at 25 cmH2O. Tissues were processed through paraffin, and
standard 5-µm sections were stained with hematoxylin and eosin for
light microscopic examination.
Statistical analysis. The effects of intrathoracic vagal denervation and sham surgery on arterial pH, blood-gas tensions, breathing patterns, pulmonary mechanics, heart rate, arterial blood pressure, and surface tension of the bronchoalveolar lavage were analyzed with the use of ANOVA for repeated measures. If a significant difference was observed, Tukey's test was performed to determine where the differences were across time but within a given group. Differences in sleep state, postoperative course, and surfactant aggregates were analyzed using Student's t-test. Regression analysis using the generalized estimating equation approach, a method analogous to ANOVA, was utilized to determine the frequency of augmented breaths and the effects of time within and between the groups. Furthermore, a generalized additive model was used to explore the possible nonlinearity of the slopes of the frequency of augmented breaths. The incidence of manual positive pressure ventilation and oxygen requirement during the postoperative course was analyzed using the Fisher's exact test. All values are given as means ± SD, and statistical significance was considered as P < 0.05.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Postoperative course.
The vagally denervated animals were significantly slower to initiate
spontaneous breathing after surgery and required manual ventilation and
supplemental oxygen to establish effective pulmonary gas exchange
compared with the sham-operated group (P < 0.05; Table 1). Similarly, sham-operated
animals, compared with the denervated animals, were significantly
faster in achieving an awake and alert state (0.5 vs. 6 h,
respectively; P < 0.05). Furthermore, all denervated
animals required additional heating via a heat lamp as rectal
temperature decreased to 36.6°C by 30 min postoperatively (Table 1).
Six of the eight denervated animals developed respiratory failure ~20
h postoperatively, as evidenced by prolonged apneic periods and
gasping. In contrast, all sham-operated lambs maintained normal
breathing patterns throughout the study course.
|
Arterial pH and blood-gas tensions.
Arterial pH and blood-gas tensions were measured while the animals were
breathing room air and are given in Table
2. Postoperatively, there were no
significant differences in PaCO2 and arterial pH between sham-operated and denervated lambs until 16 and 20 h, respectively. PaO2 was significantly lower in the
denervated than in sham-operated lambs immediately after surgery and
remained lower until the end of the study compared with the
sham-operated lambs (P < 0.05).
|
Pulmonary mechanics and breathing patterns.
Pulmonary function variables including respiratory rate, inspiratory
and expiratory times, tidal volume, minute ventilation, augmented
breaths, respiratory system and lung compliance, and respiratory system
and pulmonary resistance are given in Figs. 2-5. The respiratory
rate and inspiratory and expiratory times were similar in both groups
before surgery. However, respiratory rate was significantly reduced in
the denervated group after the immediate postoperative period compared
with the sham-operated group (P < 0.05;
Fig. 2A). Vagally denervated
animals exhibited a significant increase in inspiratory time compared
with sham-operated animals during the recovery period; however, there
were no significant differences thereafter (Fig. 2B). In
contrast, the expiratory time was consistently twofold higher in the
denervated group compared with the sham-operated animals
(P < 0.05; Fig. 2C).
|
|
|
|
|
|
Sleep states.
The three sleep states [non-rapid eye movement (NREM), rapid eye
movement (REM), and awake] are given as a percentage of total recorded
time in sham-operated and vagally denervated animals in Table
3. No significant differences existed in
the distribution of NREM and REM sleep or arousal states between the
two groups.
|
Cardiovascular variables: systolic, diastolic, and mean blood
pressures and heart rate.
No significant differences were observed between sham-operated and
vagally denervated animals in systolic, diastolic, or mean blood
pressures and heart rates (Figs. 8 and
9).
|
|
Large- and small-aggregate surfactants, surface tension, and light and electron microscopy. Phospholipid content of large- and small-aggregate surfactants were similar in both the sham-operated and the denervated groups. The phospholipid content in large-aggregate surfactant was 39 ± 21 and 35 ± 6 mg/kg in sham-operated and denervated lambs, respectively. The phospholipid levels in small-aggregate surfactant were 61 ± 36 mg/kg in the sham-operated and 58 ± 18 mg/kg in the denervated animals (P > 0.05).
Measurement of the ability of large-aggregate surfactant to reduce surface tension at an air-liquid interface using the captive bubble technique showed no significant differences between the two groups (Fig. 10). Transmission electron microscopy showed no differences in alveolar type II cell morphology, lamellar body content/structure, or tubular myelin formation in the extracellular space (Fig. 11). In addition, light microscopy showed no differences in lung architecture between the two groups (Fig. 12). Specifically, there was no evidence of inflammation or interstitial edema in the two groups. Alveolar edema could not be assessed because the lungs had been lavaged.
|
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
General. This is the first study to investigate the effects of intrathoracic vagal denervation in unanesthetized and spontaneously breathing animals during early postnatal life. We have shown that vagal denervation leads to hypoxemia and decreased pulmonary compliance. Furthermore, vagal denervation during this time leads to changes in breathing patterns, including prolongation of the duration of expiration and reductions in respiratory rate and minute ventilation. In the early postoperative period, the frequency of augmented breaths was lower in the denervated group but gradually increased and then plateaued over the course of the study. In contrast, the sham-operated group showed a negative correlation in the frequency of augmented breaths over time. Finally, we find no aberrations in the surfactant system, as evidenced by the absence of changes in biochemical and physical properties of the bronchoalveolar lavage and presence of normal type II cells. The absence of differences between sham-operated and denervated lambs in light and electron microscopy examinations and surface tension suggests that pulmonary edema is not the likely cause of hypoxemia and decreased compliance in vagally denervated animals. Thus our results indicate that postnatal vagal denervation does not directly influence the status of the lung. Rather, its primary effects relate to alteration of neural control of ventilation.
Postoperative course. In the immediate postoperative period, denervated animals required manual ventilation, supplemental oxygen, an external heating source and took longer times to be extubated compared with the sham-operated animals. This difference is most likely due to lack of afferent vagal feedback influencing the breathing pattern, ventilation, lung volume (14), and/or excitatory input provided to the inspiratory activity by the unmyelinated vagal fibers (8).
Pulmonary gas exchange. In our current study, denervated animals consistently displayed arterial hypoxemia during the postoperative period. We observed no significant differences in either arterial pH or PaCO2 until the end of the study, when vagally denervated lambs developed respiratory acidosis and hypercapnea compared with the sham-operated animals. These effects cannot be ascribed to surgical instrumentation because the sham-operated animals underwent similar surgical manipulation but remained normoxic and normocapneic throughout the study.
During the early postoperative period, vagally denervated animals exhibited an intrinsic gas-exchange problem characterized by a normal PaCO2, increased alveolar-arterial oxygen difference, and hypoxemia. Respiratory failure gradually ensued, as evidenced by increased PaCO2 and decreased PaO2 and pH near the end of the study. Although hypoxemia may arise from a number of different pathological mechanisms, the three possible mechanisms relevant to this study are low ventilation-to-perfusion ratio, right-to-left intrapulmonary shunt, or diffusion impairment. We speculate that the hypoxemia may arise from either pulmonary atelectasis or perhaps pulmonary edema, both of which would both result in a low ventilation-to-perfusion ratio and, possibly, right-to-left intrapulmonary shunt. Occurrence of these mechanisms is supported by the observed changes in the pulmonary mechanics, namely, the reduction in both lung and respiratory system compliance. Augmented breaths or sighs, defined as a biphasic spontaneous inspiratory effort, i.e., an "inspiration on inspiration," occurs in almost all mammalian species and functions to prevent pulmonary atelectasis and to increase lung compliance and contributes to intrapulmonary gas diffusion (9, 11, 34, 35, 53). Their frequency is, however, species dependent, occurring as frequently as 45 cycles per hour in adult mice vs. 3 cycles per hour in adult humans (34). Furthermore, in human infants, Cross et al. (9) and Thach and Taeusch (53) have shown that augmented breaths tend to occur more frequently on postnatal day 1 compared with days 2-5. Mead and Collier (35) and Williams et al. (56) provided evidence that, in the absence of large inflations, both lightly anesthetized spontaneously breathing and artificially ventilated animals experience a decreased pulmonary compliance. The decrease in transpulmonary pressure was directly proportional to the decrease in pulmonary compliance, and as few as four inflations restored the pulmonary compliance to the control values (56). Furthermore, postmortem examination did not show gross atelectasis among animals that received intermittent inflations (56). Studies by Larrabee and Knowlton (28) and Knowlton and Larrabee (27) provided indirect evidence that slowly adapting and rapidly adapting vagal fibers mediate the inspiratory inhibitory and excitatory responses to lung inflation, respectively. Similarly, Glogowska et al. (17) showed that vagal afferents mediated both spontaneous and cyanide-induced augmented breaths. However, Bartlett (2) showed that both vagal denervation and/or chemodenervation eliminate spontaneous deep breaths. It is important to note that hypoxia (10% O2) caused a severalfold increase in the incidence of sighs compared with normoxic conditions, suggesting a complex interaction between afferent chemoreceptor and vagally mediated inspiratory drives (2). In our current study, during the early postoperative period, augmented breaths were lower in the denervated group and, along with decreased respiratory rate and low dynamic FRC, could result in atelectasis and poor pulmonary compliance (35, 56), which gradually worsened over time. Provision of supplemental oxygen might have also exacerbated the pulmonary atelectasis. Although the frequency of augmented breaths increased in the denervated group after the initial few hours after surgery, late resurgence of such breaths might not have been able to reverse the atelectasis, resulting in persistent hypoxemia and low pulmonary compliance. In contrast, depending on the definition of augmented breaths used, sham-operated animals did not manifest a decrease in frequency until 14-18 h postoperatively despite normoxemia. A remarkable and persistent increase in the frequency of augmented breaths in the denervated group, 5 h after surgery, is likely due to hypoxemia, as shown by Bartlett (2) and Glogowska et al. (17). Although evidence from the previous studies performed in anesthetized animals suggests that the decreased or absent augmented breaths due to lack of vagal input are not restored by hypoxemia (2, 17), our data show this not to be the case. Several possible explanations for the persistence of augmented breaths in our denervated animals can be entertained. The animals in our studies were less than 24 h old and would be expected to have higher number of sighs (9) compared with the older neonates or adult subjects (45, 53). Persistence of sighs in the denervated group after vagal denervation may reflect the net effect of loss of the sigh-promoting action of vagal afferents and the addition of such an effect by hypoxia. After the immediate postoperative period, we did not correct hypoxemia with supplemental oxygen, which might be the underlying stimulatory mechanism as shown previously (2, 17). Furthermore, vagal denervation was performed in the intrathoracic region, which would leave aortic chemoreception and laryngeal afferent input intact, possibly playing a role in the production of augmented breaths. Finally, the relatively noninvasive method of recording the augmented breaths using the esophageal pressure, which correlates well with tidal volume measurements, provided us an opportunity to record breathing patterns over a prolonged period and might have mitigated the effects of acute and short-term studies (9). Further experiments are required to clarify the relative roles of anesthesia, hypoxemia, postnatal age, and the site of vagal denervation on the frequency and depth of augmented breaths. Toward the end of the study (~16 h postoperatively), vagally denervated lambs exhibited an increase in PaCO2 and a decrease in pH relative to sham-operated animals, which did not increase minute ventilation. This time period was also associated with a lowered respiratory rate and high expiratory times associated with periods of apnea in vagally denervated animals. Similar results were reported by Schwieler (50) who showed that vagal denervation in the newborn leads to decreased pH and increased PaCO2 without any effective stimulation of respiration. Another possible factor that could contribute to the observed apnea is diaphragmatic muscle fatigue as a result of increased anaerobic respiration toward the end of the study (20).Breathing patterns/pulmonary mechanics. We observed a significant reduction in respiratory rate and an increase in expiratory time, followed by irregular breathing and apneas, in vagally denervated compared with the sham-operated animals. Similar results were described in a study by Schwieler (50), in which cervical vagal denervation in newborns reduced breathing rate that became periodic or gasplike and was followed by apneic episodes (50). Increases in expiratory and inspiratory times have been shown by a number of investigators who have performed either cervical vagal denervation or vagal cooling in rabbits, cats, dogs, and newborn rats (10, 14, 46, 54).
In addition, we observed that minute ventilation was significantly lower in vagally denervated lambs by 6 h postoperatively compared with sham-operated animals. Decreased minute ventilation has also been observed in cervically vagally denervated rabbits, lambs, and rats and may be responsible for the resulting hypoxemia in our vagally denervated lambs (14, 33, 38). Previous studies have shown up to a threefold increase in tidal volume of vagally denervated rabbits and rats (newborn) compared with control animals (14, 55). However, tidal volume did not change in the denervated lambs compared with the sham-operated animals over the course of the study. The failure of tidal volume to increase may have two explanations. First, the small increase in inspiratory time would limit the inspiratory volume. Because previous studies showed large increases in inspiratory time, this could lead to an increase in tidal volume. Second, the low lung compliance in vagally denervated animals presumably offsets an increase in the driving pressure to produce a tidal volume comparable to sham-operated animals. These changes in pulmonary function data and mechanics support the hypothesis that vagal-afferent input provides the positive feedback in newborns required for the maintenance of breathing patterns, ventilation, and pulmonary mechanics. No changes were observed in either static or dynamic respiratory system resistance between the sham-operated and denervated animals. Similar results in adult rabbits were reported by Mortola et al. (38). In addition, our experiments showed that both respiratory system compliance and lung compliance were lower in vagally denervated animals compared with the sham-operated group. This may be due to the development of alveolar derecruitment (atelectasis), connective tissue abnormalities, disturbances in surfactant function, or pulmonary edema (6, 19, 25). Atelectasis would reduce lung volume history over time, depending on the breathing patterns exhibited by the subject. In our study, vagally denervated animals exhibited increased expiratory times without an elevation in inspiratory times. Together with the loss of lower airway afferent input from slowly adapting and rapidly adapting pulmonary vagal receptors to the upper airway, this pattern of breathing could result in substantial derecruitment of alveoli, which would change lung volume history over time and explain the lower lung compliance values obtained from vagally denervated animals compared with the sham-operated animals. In our present study, we have shown that dynamic FRC was maintained above the passive FRC in the sham-operated animals. In contrast, dynamic end-expiratory lung volume was similar to the passive FRC in the denervated group as determined by expiratory flow-volume curves. In newborns, evidence suggests that end-expiratory lung volume is dynamically maintained above the passive FRC (5, 36). This strategy is essential for a number of reasons, including the presence of an oxygen reserve, to minimize the energetic losses during lung expansion, and to limit the cyclic oscillations in alveolar and blood gas (5). Establishment of a dynamic FRC above the passive FRC in newborns is accomplished via expiratory braking, which serve to prolong the expiratory time constant, resulting in airway pressures more positive than during normal respiration (36, 47, 52). During expiratory braking, lung volume is above the end-expiratory volume and expiratory flow approaches the zero baseline (47). Studies have shown that expiratory braking may be attained via two separate mechanisms. One mechanism involves the upper airway and larynx, which increases the resistance of airflow during expiration by contraction of the thyroarytenoid muscles, resulting in adduction of the vocal cords (5). In newborns, Fisher et al. (15) showed that this mechanism of expiratory braking lengthens expiratory time by up to 75% during the early newborn period. The second mechanism of expiratory braking involves retardation of the expiratory flow by postinspiratory contraction of the diaphragm and other inspiratory muscles (47). Tonic activity of the diaphragm and intercostal muscles was documented at end expiration in newborns, and lung volume decreased when this activity disappeared during apneic periods (31, 40). In our present study, the animals were endotracheally intubated during pulmonary function tests, eliminating laryngeal braking. Thus braking mechanisms involving the diaphragm and intercostals were responsible for the differences in FRC of the sham-operated and vagally denervated animals. In the lamb and puppy, it has been observed that deflation of the lung whether by opening a tracheal window, thus bypassing the upper airway, or by exposing the airway to an end-expiratory subatmospheric pressure causes an increase in thyroarytenoid activity, resulting in the adduction of the vocal cords. This response reflects an attempt by the animal to maintain an elevated lung volume (42). In addition, when the upper airway is bypassed, postinspiratory diaphragmatic activity will increase in an attempt to maintain lung volume (21). We speculate that the differences in expiratory braking between the two groups would have been greater had the larynx not been intubated. Pulmonary edema in vagally denervated animals has previously been described by Berry et al. (4). Furthermore, pulmonary edema could lead to dysfunction of the surfactant system, which, in turn, could be responsible for the observed changes in arterial pH and blood-gas tensions. However, in our current study, vagally denervated animals showed no differences in phospholipid content of large- or small-aggregate surfactants compared with sham-operated animals, indicating that total phospholipid levels remained intact in vagally denervated animals. In addition, electron micrographs of tissue samples taken from the right middle lobes of vagally denervated and sham-operated animals depict the presence of tubular myelin formation and secretion of surfactant from lamellar bodies, indicating that secretion of surfactant phospholipids and assembly of tubular myelin from phospholipids and surfactant-associated proteins was preserved in vagally denervated animals. To quantify surfactant function, we measured surface tension-lowering properties of the bronchoalveolar lavage using the captive bubble technique of Schürch et al. (49) and observed no difference in large-aggregate surface tension-lowering properties between the sham-operated and vagally denervated lambs, indicating that the surfactant produced was fully capable of reducing surface tension and promoting stability at the alveolar interface (49).Thermoregulation. Although the primary strategy adopted by adults to increase alveolar ventilation is hyperpnea, many newborn species respond to hypoxemia predominantly with hypometabolism as opposed to hyperpnea (37, 51). Hypoxic reduction in metabolic rate is also supported by the observation that denervated animals were unable to maintain normal body temperatures over the postoperative period and had to be warmed using a heat lamp. Hypoxia decreases body temperature in a number of animal species (16, 39, 44, 57) via a decrease in thermogenesis, although metabolic and ventilatory responses vary depending on species and postnatal age and weight (16).
Sleep states. We observed no significant differences in sleep states between the vagally denervated and sham-operated lambs over the 24-h study course; thus the observed decreases in breathing frequency, arterial gas tensions, and pH cannot be explained on the basis of deficiency or excess of one particular sleep state. The prevalence of various sleep states in our denervated and sham-operated animals may not represent the normal (physiological) distribution due to the surgical procedures. However, the lack of difference in the distribution of sleep states between the two groups suggests that the respiratory failure was not sleep dependent.
Cardiovascular variables. Vagal innervation influences heart rate and contractility via a parasympathetic mechanism. To avoid the cardiac effects of vagal denervation, we minimized trauma to the vagal fibers, leading to the cardiac plexus, and observed no significant differences between sham-operated and denervated animals in heart rate, systolic pressure, or diastolic pressure. Absence of changes in postnatal pulmonary blood flow with antenatal intrathoracic vagal denervation has also been previously established by Hasan et al. (22).
In summary, we report that vagally denervated neonatal lambs developed hypoxemia and respiratory failure as well as lower minute ventilation and breathing rate compared with the sham-operated group. Furthermore, in the denervated animals, respiratory system and lung compliance were reduced without any pulmonary histological changes or aberrations in the surfactant system. The results suggest that vagal innervation is necessary for the maintenance of normal breathing patterns and pulmonary gas exchange during the early neonatal period. Because the FRC in the neonates is determined dynamically, it is likely that the deleterious effects of intrathoracic vagal denervation on pulmonary mechanics and gas exchange during the early newborn period are due to a progressive atelectasis secondary to decreased respiratory frequency and reduced end-expiratory lung volume. Furthermore, a deficiency in the frequency of augmented breaths during the early postoperative period in the denervated animals likely exacerbated the progressive atelectasis.| |
ACKNOWLEDGEMENTS |
|---|
We acknowledge the statistical consultation provided by Dr. Gordon Fick, the technical assistance of Ather Bano, Helena Frndova, Anita Rigaux, Stanley Cheng, and the secretarial support of Marion Taggart.
| |
FOOTNOTES |
|---|
Financial support was provided by the Medical Research Council of Canada, the Alberta Heritage Foundation for Medical Research, and in part by the Alberta Children's Hospital Foundation.
Address for reprint requests and other correspondence: S. U. Hasan, Dept. of Pediatrics, Respiratory Research Group, Faculty of Medicine, The Univ. of Calgary, 3330 Hospital Dr. N.W., Calgary, Alberta, Canada T2N 4N1 (E-mail: hasans{at}ucalgary.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.
Received 24 January 2001; accepted in final form 21 June 2001.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Alcorn, D,
Adamson TM,
Maloney JE,
and
Robinson PM.
Morphological effects of chronic bilateral phrenectomy or vagotomy in the fetal lamb lung.
J Anat
130:
683-695,
1980[Web of Science][Medline].
2.
Bartlett, D, Jr.
Origin and regulation of spontaneous deep breaths.
Respir Physiol
12:
230-238,
1971[Web of Science][Medline].
3.
Bartlett, GR.
Phosphorus assay in column chromatography.
J Biol Chem
234:
466-468,
1959
4.
Berry, D,
Ikegami M,
and
Jobe A.
Respiratory distress and surfactant inhibition following vagotomy in rabbits.
J Appl Physiol
61:
1741-1748,
1986
5.
Bryan, AC,
and
England SJ.
Maintenance of an elevated FRC in the newborn. Paradox of REM sleep.
Am Rev Respir Dis
129:
209-210,
1984[Web of Science][Medline].
6.
Comroe, JH.
Mechanical factors in breathing.
In: Physiology of Respiration. New York: Year Book Medical, 1974, p. 94-141.
7.
Coombs, HC,
and
Pike FH.
The nervous control of respiration in kittens.
Am J Physiol
95:
681-693,
1930.
8.
Cross, BA,
Jones PW,
and
Guz A.
The role of vagal afferent information during inspiration in determining phrenic motoneurone output.
Respir Physiol
39:
149-167,
1980[Web of Science][Medline].
9.
Cross, KW,
Klaus M,
Tooley WH,
and
Weisser K.
The response of the new-born baby to inflation of the lungs.
J Physiol (Lond)
151:
551-565,
1960.
10.
Davenport, PW,
Sant'Ambrogio FB,
and
Sant'Ambrogio G.
Adaptation of tracheal stretch receptors.
Respir Physiol
44:
339-349,
1981[Web of Science][Medline].
11.
Davis, GM,
and
Moscato J.
Changes in lung mechanics following sighs in premature newborns without lung disease.
Pediatr Pulmonol
17:
26-30,
1994[Web of Science][Medline].
12.
Delacourt, C,
Canet E,
Praud J-P,
and
Bureau MA.
Influence of vagal afferents on diphasic ventilatory response to hypoxia in newborn lambs.
Respir Physiol
99:
29-39,
1995[Web of Science][Medline].
13.
Duron, B,
and
Marlot D.
Nervous control of breathing during postnatal development in the kitten.
Sleep
3:
323-330,
1980[Web of Science][Medline].
14.
Fedorko, L,
Kelly EN,
and
England SJ.
Importance of vagal afferents in determining ventilation in newborn rats.
J Appl Physiol
65:
1033-1039,
1988
15.
Fisher, JT,
Mortola JP,
Smith JB,
Fox GS,
and
Weeks S.
Respiration in newborns: development of the control of breathing.
Am Rev Respir Dis
125:
650-657,
1982[Web of Science][Medline].
16.
Frappell, P,
Lanthier C,
Baudinette RV,
and
Mortola JP.
Metabolism and ventilation in acute hypoxia: a comparative analysis in small mammalian species.
Am J Physiol Regulatory Integrative Comp Physiol
262:
R1040-R1046,
1992
17.
Glogowska, M,
Richardson PS,
Widdicombe JG,
and
Winning AJ.
The role of the vagus nerves, peripheral chemoreceptors and other afferent pathways in the genesis of augmented breaths in cats and rabbits.
Respir Physiol
16:
179-196,
1972[Web of Science][Medline].
18.
Gluckman, PD,
Sizonenko SV,
and
Bassett NS.
The transition from fetus to neonate
an endocrine perspective.
Acta Paediatr Suppl
88:
7-11,
1999[Medline].
19.
Greaves, IA,
Hildebrandt J,
and
Hoppin FG, Jr.
Micromechanics of the lung.
In: Handbook of Physiology. The Respiratory System. Mechanics of Breathing. Bethesda, MD: Am. Physiol. Soc, 1986, sect. 3, vol. III, pt. 1, chapt. 14, p. 217-232.
20.
Haddad, GG,
and
Mellins RB.
Hypoxia and respiratory control in early life.
Ann Rev Physiol
46:
629-643,
1984[Web of Science][Medline].
21.
Harding, R.
State-related and developmental changes in laryngeal function.
Sleep
3:
307-322,
1980[Web of Science][Medline].
22.
Hasan, SU,
Bano A,
and
Rigaux A.
Cardiovascular responses during transition from fetal to neonatal life in intact and vagotomized lambs (Abstract).
Pediatr Res
43:
284A,
1998.
23.
Hasan, SU,
and
Rigaux A.
Arterial oxygen tension threshold range for the onset of arousal and breathing in fetal sheep.
Pediatr Res
32:
342-349,
1992[Web of Science][Medline].
24.
Hasan, SU,
and
Rigaux A.
Effect of bilateral vagotomy on oxygenation, arousal, and breathing movements in fetal sheep.
J Appl Physiol
73:
1402-1412,
1992
25.
Hoppin, FG,
and
Hildebrandt J.
Mechanical properties of the lung.
In: Bioengineering Aspects of the Lung, edited by West JB.. New York: Marcel Dekker, 1977, p. 83-162.
26.
Jobe, AH.
Surfactant in the perinatal period.
Early Hum Dev
29:
57-62,
1992[Web of Science][Medline].
27.
Knowlton, GC,
and
Larrabee MG.
A unitary analysis of pulmonary volume receptors.
Am J Physiol
147:
100-114,
1946.
28.
Larrabee, MG,
and
Knowlton GC.
Excitation and inhibition of phrenic motoneurons by inflation of the lungs.
Am J Physiol
47:
90-99,
1946.
29.
Lesouef, PN,
England SJ,
and
Bryan AC.
Passive respiratory mechanics in newborns and children.
Am Rev Respir Dis
129:
552-556,
1984[Web of Science][Medline].
30.
Lijowska, AS,
Reed NW,
Chiodini BA,
and
Thach BT.
Sequential arousal and airway-defensive behavior of infants in asphyxial sleep environments.
J Appl Physiol
83:
219-228,
1997
31.
Lopes, J,
Muller NL,
Bryan MH,
and
Bryan AC.
Importance of inspiratory muscle tone in maintenance of FRC in the newborn.
J Appl Physiol
51:
830-834,
1981
32.
Marlot, D,
and
Duron B.
Postnatal development of vagal control of breathing in the kitten.
J Physiol (Lond)
75:
891-900,
1979.
33.
Marsland, DW,
Callahan BJ,
and
Shannon DC.
The afferent vagus and regulation of breathing in response to inhaled CO2 in awake newborn lambs.
Biol Neonate
27:
102-107,
1975[Web of Science][Medline].
34.
McCutcheon, FH.
Atmospheric respiration and the complex cycles in mammalian breathing mechanisms.
J Cell Comp Physiol
41:
291-303,
1953.
35.
Mead, J,
and
Collier C.
Relation of volume history of lungs to respiratory mechanics in anesthetized dogs.
J Appl Physiol
14:
669-678,
1959
36.
Mortola, JP.
Dynamics of breathing in newborn mammals.
Physiol Rev
67:
187-243,
1987
37.
Mortola, JP.
How newborn mammals cope with hypoxia.
Respir Physiol
116:
95-103,
1999[Web of Science][Medline].
38.
Mortola, JP,
Fisher JT,
and
Sant'Ambrogio G.
Vagal control of the breathing pattern and respiratory mechanics in the adult and newborn rabbit.
Pflügers Arch
401:
281-286,
1984[Web of Science][Medline].
39.
Mortola, JP,
Rezzonico R,
and
Lanthier C.
Ventilation and oxygen consumption during acute hypoxia in newborn mammals: a comparative analysis.
Respir Physiol
78:
31-43,
1989[Web of Science][Medline].
40.
Olinsky, A,
Bryan MH,
and
Bryan AC.
Influence of lung inflation on respiratory control in neonates.
J Appl Physiol
36:
426-429,
1974
41.
Oliver, TK,
Demis JA,
and
Bates GD.
Serial blood gas tensions and acid-base balance during the first hour of life in human infants.
Acta Paediatr
50:
346-360,
1961[Medline].
42.
Oommen, MP,
and
Sant'Ambrogio G.
Respiratory Functions of the Upper Airway. New York: Marcel Dekker, 1988, p. 1-645.
43.
Oyarzun, MJ,
and
Clements JA.
Control of lung surfactant by ventilation, adrenergic mediators, and prostaglandins in the rabbit.
Am Rev Respir Dis
117:
879-891,
1978[Web of Science][Medline].
44.
Pedraz, C,
and
Mortola JP.
CO2 production, body temperature, and ventilation in hypoxic newborn cats and dogs before and after body warming.
Pediatr Res
30:
165-169,
1991[Web of Science][Medline].
45.
Perez-Padilla, R,
West P,
and
Kryger MH.
Sighs during sleep in adult humans.
Sleep
6:
234-243,
1983[Web of Science][Medline].
46.
Pisarri, TE,
Yu J,
Coleridge HM,
and
Coleridge JCG
Background activity in pulmonary vagal C-fibers and its effects on breathing.
Respir Physiol
64:
29-43,
1986[Web of Science][Medline].
47.
Remmers, JE,
and
Bartlett D, Jr.
Reflex control of expiratory airflow and duration.
J Appl Physiol
42:
80-87,
1977
48.
Rigatto, H,
Hasan SU,
Jansen A,
Gibson D,
Nowaczyk B,
and
Cates D.
The effect of total peripheral chemodenervation on fetal breathing and on the establishment of breathing at birth in sheep.
In: Fetal and Neonatal Development, edited by Jones CT.. Ithaca, NY: Perinatology Press, 1988, p. 613-621. (Research in Perinatal Medicine VII)
49.
Schürch, S,
Bachofen H,
Goerke J,
and
Green F.
Surface properties of rat pulmonary surfactant studied with the captive bubble method: absorption, hysteresis, stability.
Biochim Biophys Acta
1103:
127-136,
1992[Medline].
50.
Schwieler, GH.
Respiratory regulation during postnatal development in cats and rabbits and some of its morphological substrate.
Acta Physiol Scand
304:
7-123,
1968.
51.
Stainsby, WN,
and
Otis AB.
Blood flow, blood oxygen tension, oxygen uptake, and oxygen transport in skeletal muscle.
Am J Physiol
206:
858-866,
1964.
52.
Thach, BT,
Abroms IF,
Frantz ID,
Sotrel A, III,
Bruce EN,
and
Goldman MD.
Intercostal muscle reflexes and sleep breathing patterns in the human infant.
J Appl Physiol
48:
139-146,
1980
53.
Thach, BT,
and
Taeusch HW, Jr.
Sighing in newborn human infants: role of inflation-augmenting reflex.
J Appl Physiol
41:
502-507,
1976
54.
Trenchard, D.
Role of pulmonary stretch receptors during breathing in rabbits.
Respir Physiol
29:
231-246,
1977[Web of Science][Medline].
55.
Trippenbach, T,
Kelly G,
and
Marlot D.
Effects of tonic vagal input on breathing pattern in newborn rabbits.
J Appl Physiol
59:
223-228,
1985
56.
Williams, JV,
Tierney DF,
and
Parker HR.
Surface forces in the lung, atelectasis, and transpulmonary pressure.
J Appl Physiol
21:
819-827,
1966
57.
Wong, KA,
Bano A,
Rigaux A,
Wang B,
Bharadwaj B,
Schurch S,
Green F,
Remmers JE,
and
Hasan SU.
Pulmonary vagal innervation is required to establish adequate alveolar ventilation in the newborn lamb.
J Appl Physiol
85:
849-859,
1998
This article has been cited by other articles:
|
|
 |
|
  L. Gahlot, F. H. Y. Green, A. Rigaux, J. M. Schneider, and S. U. Hasan Role of vagal innervation on pulmonary surfactant system during fetal development J Appl Physiol, May 1, 2009; 106(5): 1641 - 1649. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Roy, N. Samson, F. Moreau-Bussiere, A. Ouimet, D. Dorion, S. Mayer, and J.-P. Praud Mechanisms of active laryngeal closure during noninvasive intermittent positive pressure ventilation in nonsedated lambs J Appl Physiol, November 1, 2008; 105(5): 1406 - 1412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Sindelar, A. Jonzon, A. Schulze, and G. Sedin Surfactant replacement partially restores the activity of pulmonary stretch receptors in surfactant-depleted cats J Appl Physiol, February 1, 2006; 100(2): 594 - 601. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Arsenault, F. Moreau-Bussiere, P. Reix, T. Niyonsenga, and J.-P. Praud Postnatal maturation of vagal respiratory reflexes in preterm and full-term lambs J Appl Physiol, May 1, 2003; 94(5): 1978 - 1986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Lalani, J. E. Remmers, Y. MacKinnon, G. T. Ford, and S. U. Hasan Hypoxemia and low Crs in vagally denervated lambs result from reduced lung volume and not pulmonary edema J Appl Physiol, August 1, 2002; 93(2): 601 - 610. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Thach Fast breaths, slow breaths, small breaths, big breaths: importance of vagal innervation in the newborn lung J Appl Physiol, November 1, 2001; 91(5): 2298 - 2300. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
FRC) in sham-operated and vagally denervated animals indicates
that sham-operated animals maintained a significantly higher 
