Vol. 91, Issue 5, 2301-2313, November 2001
INVITED EDITORIAL
Effects of vagal denervation on cardiorespiratory and
behavioral responses in the newborn lamb
Salim
Lalani,
John E.
Remmers,
Francis H.
Green,
Ashfaq
Bukhari,
Gordon T.
Ford, and
Shabih U.
Hasan
Department of Pediatrics, Respiratory Research Group, Faculty of
Medicine, The University of Calgary, Calgary, Alberta, Canada T2N
4N1
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ABSTRACT |
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
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INTRODUCTION |
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.
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METHODS |
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).
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Fig. 1.
Experimental design. Surgery was performed within 24 h of
birth, and the animals were studied for a period of ~24 h
postoperatively. During the course of the study, sleep states,
diaphragmatic electromyogram (EMGdia), augmented
breaths, arterial blood pressure (BP), heart rate (HR), esophageal
pressure (PESO), and rectal temperature were continuously
recorded. Pulmonary function tests were performed 1 h
preoperatively and during the recovery period, 6 and 24 h
postoperatively. Pulmonary function testing included measurements of
respiratory rate (f), inspiratory time (TI), expiratory
time (TE), tidal volume (VT), minute
ventilation (MV), compliance (C), and resistance (R). Arterial pH and
blood-gas tensions were measured at least every 2 h or more
frequently if clinically indicated. After lambs were euthanized, the
right middle lobe (RML) was sectioned and a bronchoalveolar lavage
(BAL) was performed on the remaining right and left lobes.
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Dextrose (10%) in normal saline was continuously infused intravenously
at 90-120
ml · kg
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.
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RESULTS |
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).
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Fig. 2.
Respiratory rate, inspiratory time, and expiratory time.
A: respiratory rate (per minute) significantly decreased in
vagally denervated animals by 6 and 24 h postoperatively compared
with the sham-operated group. B: no significant differences
were observed in inspiratory time except during the recovery period in
the denervated animals. C: expiratory time was significantly
higher in the denervated lambs during recovery and 6 and 24 h
postoperatively compared with the sham-operated group.
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Fig. 3.
Pulmonary function variables: VT/kg
(A) and MV/kg (B). A: no differences
were observed in VT/kg between sham-operated and vagally
denervated animals. B: MV/kg
(ml · min1 · kg1) was
significantly lower in vagally denervated animals at 6 and 24 h
postoperatively compared with sham-operated animals.
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Fig. 4.
Augmented breaths. A: frequency of augmented
breaths, defined as an inspiration on inspiration and at least 50%
increase in esophageal pressure and EMGdia compared with
the preceding 10 breaths. The frequency of the augmented breaths
decreased over time in the sham-operated group (8.41 0.30 × time; P = 0.001). In contrast, a significant
increase in the slope of the augmented breaths [(8.41 5.13) + (0.54 0.30)] × time was observed in the
denervated group (P = 0.001; A).
B: frequency of augmented breaths, defined as an inspiration
on inspiration and a twofold increase in esophageal pressure compared
with the preceding 10 breaths. The sham-operated group showed a
decrease in the frequency of the augmented breaths over time (5.97 0.20 × time; P = 0.001). In contrast, a
significant increase in the slope of the augmented breaths [(5.97 4.22) + (0.32 0.20) × time; P = 0.001] was observed in the denervated group. The differences in the
direction of the slopes using both definitions (A and
B) was also significantly different between the 2 groups
over the duration of the study (P < 0.001).
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Fig. 5.
Pulmonary function variables:
compliance and resistance. Static respiratory system compliance per
body weight (CRS/kg; A) and dynamic lung
compliance per body weight (CDYN/kg; B) were
significantly lower in vagally denervated animals compared with
sham-operated animals at 6 and 24 h postoperatively. In contrast,
dynamic pulmonary resistance (RDYN; D) and
static respiratory system resistance (RRS; C)
were similar in sham-operated and vagally denervated animals.
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The tidal volume was similar between the sham-operated and the vagally
denervated animals both before and after surgery (Fig. 3A). In contrast, minute
ventilation significantly decreased in the denervated group compared
with the sham-operated lambs at 6 and 24 h postoperatively
(P < 0.05; Fig. 3B).
The frequency of augmented breaths is presented in Fig.
4 in five 4-hour postoperative time bins
(2-5, 6-9, 10-13, 14-17, and 18-21 h). The
first hour could not be included because various data acquisition
apparatuses were still being connected to record physiological
variables from both groups and vagally denervated animals were still
requiring assisted ventilation. Similarly, only two vagally denervated
animals were alive more than 21 h after surgery, and a statistical
analysis of the data from these two animals would not be appropriate.
The generalized additive model showed no evidence of nonlinearity
in the sham-operated group. However, the relationship with time was
more complex in the denervated group, showing evidence of nonlinearity
(P = 0.0013). The frequency of augmented breaths showed
a negative correlation (P = 0.001) over time in the
sham-operated group, whereas the reverse was true for the denervated
group (P = 0.001). The degree of linearity and negative
correlation in the sham-operated group and the nonlinearity and
positive correlation in the denervated group was true for both
definitions of the augmented breaths (P = 0.001).
Furthermore, there was a significant difference in the frequency of
augmented breaths over time between the sham-operated and denervated
groups (P < 0.001; Fig. 4).
Respiratory system and pulmonary resistance using static and dynamic
methods, respectively, exhibited no significant differences between
sham-operated and vagally denervated animals at any time periods after
surgery (Fig. 5, C and
D). Lung and respiratory system compliances were
significantly lower in vagally denervated animals compared with the
sham-operated animals at 6 and 24 h after surgery
(P < 0.05; Fig. 5, A and
B).
Representative flow-volume graphs from the sham-operated and vagally
denervated animals are shown in Figs. 6
and 7. The sham-operated animal
maintained the dynamic FRC to be ~50 ml above the passive FRC (Fig.
6A). In contrast, the dynamic FRC was equivalent to the
passive FRC in the denervated animal (Fig. 6B). Overall, the sham-operated animals interrupted their expiration before reaching the
"zero" flow, whereas the denervated animals emptied the lungs right
to the passive FRC (P = 0.02; Fig. 7).
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Fig. 6.
Expiratory flow-volume curve. Flow-volume curves of
a sham-operated (A) and a vagally denervated (B)
animal with identical body weights indicate that the sham-operated
animal interrupted expiration (the "knee") before reaching passive
functional residual capacity (FRC). The dynamic FRC is 50 ml above
the passive FRC, as calculated by subtracting the real expired volume
(67 ml) from the extrapolated volume (117 ml) in the sham-operated
animal (A). In contrast, the vagally denervated animal
exhibits no difference between the dynamic and passive FRC, indicating
that expiration continues until passive FRC (B).
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Fig. 7.
Comparison of the difference between dynamic and passive
FRC ( FRC) in sham-operated and vagally denervated animals indicates
that sham-operated animals maintained a significantly higher FRC
compared with the denervated group (*P = 0.02).
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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.
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Table 3.
Distribution of various sleep states as a percentage of total time in
sham-operated and vagally denervated animals
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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).
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Fig. 8.
Systolic (A), mean (B), and
diastolic (C) blood pressure. Arterial blood pressure data
were obtained at various time bins postoperatively up to 24 h.
There were no significant differences in systolic, mean, or diastolic
blood pressures between sham-operated and vagally denervated animals.
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Fig. 9.
Heart rate analysis of sham-operated and vagally
denervated animals at various time bins postoperatively shows no
significant differences between the 2 groups at any of the time bins
postoperatively.
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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.
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Fig. 10.
Analysis of surface tension. No differences were
observed between the 2 study groups in the ability of large-aggregate
surfactants to reduce surface tension (mN/m) at an air-liquid interface
over a period of 300 s.
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Fig. 11.
Transmission electron micrographs of right middle lobes of
sham-operated (A) and vagally denervated (B)
animals. The electron micrographs show lamellar bodies (LB) and tubular
myelin (TM) within the extracellular space. In both groups, numerous
lamellar bodies were observed in the process of unfolding to form
tubular myelin. The structure of LB and TM appeared normal in both
groups.
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Fig. 12.
Light micrographs of vagally denervated (A) and
sham-operated (B) animals. Light microscopy of lung
parenchyma and airways of sham-operated and vagally denervated animals
showed no evidence of perivascular pulmonary edema, vascular
congestion, or inflammation in either group.
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DISCUSSION |
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.
| |
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J Appl Physiol
59:
223-228,
1985[Abstract/Free Full Text].
56.
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