Departments of Neurobiology and Physiological Science, Systems Neurobiology Laboratory, University of California, Los Angeles, California 90095-1527
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ABSTRACT |
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Mellen, Nicholas M. and Jack L. Feldman. Phasic Lung Inflation Shortens Inspiration and Respiratory Period in the Lung-Attached Neonate Rat Brain Stem Spinal Cord. J. Neurophysiol. 83: 3165-3168, 2000. In intact mammals, lung inflation during inspiration terminates inspiration (Breuer-Hering inspiratory reflex, BHI) and the presence of lung afferents increases respiratory frequency. To test whether these responses could be obtained in vitro, a neonate rat brain stem/spinal cord preparation retaining the lungs and their vagal innervation was used. It was found that 1) the BHI could be replicated in vitro, 2) phasic lung inflation during inspiration caused increased respiratory frequency with declining efficacy as inflation delay increased, and 3) increased respiratory frequency did not require inspiratory shortening.
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INTRODUCTION |
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In mammals, feedback from slowly adapting
pulmonary stretch receptors (SARs) modulates respiratory frequency:
lung inflation during expiration lengthens expiration (Breuer-Hering
expiratory reflex, BHE) and lung inflation during inspiration shortens
inspiration (Breuer-Hering inspiratory reflex, BHI) (Widdicombe
1986). The role of SAR feedback is particularly marked in
neonates (Fedorko et al. 1988
). In vivo, the
inspiration-terminating, expiration-promoting BH reflexes are
accompanied by central adaptation mechanisms that promote inspiration
and shorten expiration (Younes, & Polacheck 1985
). The
observation that respiratory frequency decreases by 40% in vivo after
vagotomy may be due to down-regulation of these adaptative mechanisms
(Widdicombe 1986
).
A variety of in vitro medullary preparations producing motor
output qualitatively similar to eupnea in vivo have been developed to
study the neural substrate for respiratory rhythmogenesis (Smith and Feldman 1987; Suzue 1984
). If the in vitro
preparation reproduces in vivo responses to afferent input, its
usefulness as a model for eupnea is bolstered. To test whether the
circuitry mediating SAR modulation of respiratory frequency is retained
in vitro, we used a modified neonate rat brain stem/spinal cord
preparation retaining the lungs and their vagal innervation. In this
preparation, the BHE can be replicated by inflating the lungs in
midexpiration (Mellen and Feldman 1997a
;
Murakoshi and Otsuka 1985
).
In the experiments described here, we tested whether
inspiration-triggered phasic lung inflation shortened inspiration and increased respiratory frequency consistent with in vivo observations. Both responses were replicated in vitro, validating this preparation as
a model for the study of respiratory rhythmogenesis and control. Preliminary results have appeared in abstract form (Mellen and Feldman 1997b).
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METHODS |
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General methods
Ten neonatal Sprague-Dawley rats (postnatal day 0-2) were used.
Rat pups were anesthetized by hypothermia and decerebrated just rostral
to the vagus nerve (X), which corresponds to transection through the
facial nucleus. The preparation was then transferred to a bath
consisting of (in mM) 113.0 NaCl, 3.0 KCl, 1.5 CaCl2, 1.0 MgCl2, 30.0 NaHCO3, 1.2 NaH2PO4, and 30.0 glucose
and equilibrated with 95% O2/5%
CO2 at 27°C (pH 7.4). The bath was continuously perfused with artificial cerebrospinal fluid (ACSF). The brain stem,
connected to the lungs and heart by the intact X nerve, was isolated
(see Mellen and Feldman 1997a and Fig.
1A). Because an animal's
respiratory efforts continued while it was submerged in ACSF,
the lungs did not collapse after the thorax was opened. The trachea was
cannulated (22 gauge) and connected to a syringe pump (Carnegie Medecin
M100) that was used to inflate the lungs with ACSF. Viability of the
vagal afferent pathway was tested with sustained midexpiratory
inflation (pressure 2-5 mm H2O), which elicited
expiratory lengthening. Inadvertent overinflation eliminated the
response to subsequent inflations.
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Respiratory activity from the spinal C2 ventral root was recorded using
an ACSF-filled glass electrode (100 k), amplified ×70,000,
band-pass filtered (100 Hz-3 kHz), digitized at 20 kHz, and written to
computer disk.
Rectified and integrated C2 activity (
10 ms) triggered transient
lung inflation at various delays (50-2000 ms, see Experimental protocol) after inspiratory burst onset. Inflation lasted
200-400 ms and was followed immediately by matching deflation, which
returned the lungs to their resting volume. Injectate volumes were
0.2-0.4 ml, which caused pressure changes of 2-5 mm
H2O as measured using a manometer attached
to the cannula. Data acquisition and instrumentation control
was implemented in LabView (Austin, TX).
Experimental protocol
Experiments were divided into bouts of 20-40 consecutive cycles. Respiratory periods in bouts without inflation (control) were compared with those in bouts with transient inflation applied at 50-2000 ms delays from inspiratory onset (test; Fig. 1B). Within each test bout, delay was fixed and 20-40 respiratory cycles were collected followed by at least 20 control cycles. Five delay steps (50, 500, 1000, 1500, and 2000 ms) were presented in randomized order within a given experiment. Not all delays were presented in each experiment.
Data analysis
Inspiratory bursts were rectified, integrated, and averaged in a 3000-ms window triggered at inspiratory burst onset with a 1000-ms pretrigger. For each experiment, the effect of inflation on inspiratory duration was measured. Duration was estimated by setting a threshold at half the maximal control burst amplitude and measuring the amount of time the inspiratory burst was above threshold (Fig. 1C). The null hypothesis that control and test burst durations were equal was tested using a paired t-test (Origin, Northampton, MA) on bout means pooled across experiments.
Comparison of respiratory periods with and without lung inflation was obtained using a mixed effect one-way analysis of variance (ANOVA) model on bout means (mixed effect ANOVA) where the fixed effect was the onset delay of lung inflation (control, test, test + delay, with delays of 50, 500, 1000, 1500, and 2000 ms) and the random effect was bout. The Fisher-Tukey criterion was used to test the significance of post hoc t-tests within the ANOVA model. The SAS procedure MIXED (Statistical Analysis System Institute, Cary, NC) was used for the analysis.
If respiratory frequency increase only occurred with inspiratory shortening, then test bouts with periods significantly shorter than controls would also have burst durations significantly shorter than controls. This was tested by comparing burst durations and periods of control bouts with those of test bouts with delays of 50 and 1000 ms.
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RESULTS |
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Five delay steps (50, 500, 1000, 1500, and 2000 ms) were presented
in randomized order, but of those steps only delays of 50 ms gave rise
to test inspiratory bursts significantly shorter than control bursts
[P 0.01, n = 10; mean test
duration = 515 ± 51 ms (SE); mean control
duration = 624 ± 74 ms; Fig. 1D). Test burst
durations were shortened by 18% relative to control bursts.
Respiratory frequency.
Inflation at delays <2000 ms significantly shortened the respiratory period (P < 0.05, n = 10; Table 1 and Fig. 2A). The effects of inflation varied with delay: inflation at 50 ms gave rise to respiratory periods significantly shorter than all other conditions and for all test conditions other than 500 and 1000 ms delay, periods associated with each delay were significantly different from all others (P < 0.05; Fig. 2B). At delays of 2000 ms, inflation had no effect on frequency.
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Increased respiratory frequency did not require inspiratory shortening. Bouts with inflation delays of 50 and 1000 ms both had periods significantly shorter than control (Fig. 3B, diagonal bar). Inflation at 50 ms delay significantly shortened inspiratory bursts (P < 0.05) but inflation at 1000 ms did not (P > 0.5, n = 5; Fig. 3, A and B).
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DISCUSSION |
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The present experiments tested whether phasic lung inflation triggered by inspiratory onset modulated respiratory frequency and pattern in vitro in a manner consistent with observations in vivo. We conclude that 1) transient lung inflation within the physiological range during inspiration reduces inspiratory burst duration, which is congruent with BHI in vivo; 2) inspiration-triggered phasic inflation increases respiratory frequency, which is consistent with the observation that vagotomy decreases respiratory frequency in vivo; 3) phasic inflation increases respiratory frequency with declining efficacy up to 1500 ms after inspiratory onset; and 4) inflation-induced inspiratory shortening is not required for increased respiratory frequency.
Lung inflation was used in the present experiments to ensure
selective activation of the slowly adapting mechanoreceptors that
mediate the Breuer-Hering reflexes (Widdicombe 1986).
Several lines of evidence suggest that the observed responses were
elicited by SAR activation: 1) applied pressures were at the
low end of the physiological range (Milsom 1989
);
2) the response to phasic inflation persisted for as long as
the preparation remained active; and 3) in the present
experiments, the effect of transient inflation depended on onset delay.
In an earlier study (Mellen and Feldman 1997a
),
sustained inflation at similar pressures during expiration lengthened
expiration but did not change baseline frequency in subsequent control
cycles (BHE). As a consequence, it is unlikely that the increased
respiratory frequency in response to inflation during or shortly after
inspiration is caused by nonspecific activation of lung afferents
ensuing from traumatic pressure changes. Because in vitro responses to
both sustained and transient inflation match responses in vivo, we
conclude that the neural substrate sufficient for the Breuer-Hering
reflexes is retained in the in vitro medullary preparation.
The inspiration-inhibiting, expiration-prolonging effects of inflation
associated with the classical Breuer-Hering reflex has been replicated
in vitro. In addition, the increase in respiratory frequency
accompanying phasic inflation indicates that SAR afferent activation
has a facilitatory effect on rhythm-generating circuits. This response
has been characterized in vivo (Younes and Polacheck 1985) and has been ascribed to pontine structures
(Younes and Polacheck 1981
) that were not present in our
in vitro preparation.
The observation that phasic inflation at a 1000-ms delay shortened the
respiratory period but not the inspiratory burst duration (Fig. 3,
A and B) is consistent with earlier observations
in vivo (Bartoli et al. 1973) and indicates that early
inspiratory termination is not required for shortening the subsequent
respiratory period. Because the effect of inflation on the respiratory
period declines with delay from the inspiratory burst onset,
rhythmogenic circuits are particularly sensitive to afferent inputs
during and just after inspiration.
Although in vitro medullary preparations are extensively used to study
the neural substrate for respiratory rhythmogenesis, the relationship
between rhythmic motor output in vitro and eupnea in vivo is
unresolved. It is important to note that persistence of the
Breuer-Hering reflex has been proposed as an indicator of return to
eupneic activity after ischemic insult (Pluta and Romaniuk
1990). Thus, our findings bolster the view that the
rhythmogenic and frequency-regulating circuits that are functional in
the medulla in vitro reflect those underlying eupnea in vivo.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-40959 and HL-37941 and by American Lung Association Research Grant RG-105-N.
We thank J. Gornbein of the Dept. of Biostatistics, UCLA, for help with statistical analyses.
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FOOTNOTES |
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Address for reprint requests: N. M. Mellen, Dept. of Neurobiology, Box 951763, University of California, Los Angeles, CA 90095-1763.
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 3 November 1999; accepted in final form 20 January 2000.
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REFERENCES |
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