Phasic Lung Inflation Shortens Inspiration and Respiratory Period in the Lung-Attached Neonate Rat Brain Stem Spinal Cord

Nicholas M. Mellen and Jack L. Feldman

Departments of Neurobiology and Physiological Science, Systems Neurobiology Laboratory, University of California, Los Angeles, California 90095-1527


    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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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).


    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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Fig. 1. A: schematic of lung-attached in-vitro brain stem-spinal cord. Population motor output is recorded from C2 and inspiratory onset triggers lung (L) inflation via a cannula inserted in the trachea (arrow). Afferent feedback is conveyed via the vagus nerve (X). B: test cycle protocol for delay = 50 ms: lung inflation (gray rectangles) followed by deflation to resting lung volume is applied 50 ms after inspiratory onset as detected from the rectified integrated motor output (C2). C: inflation during inspiration shortens inspiration. Mean rectified integrated bursts without (solid line) and with (50 ms delay, solid gray area) lung inflation from a single experiment (n = 11). Control (diagonal area) and 50 ms delay test (crosshatched area) burst durations are shown. D: inflation with 50 ms delay significantly shortens inspiratory burst duration (P < 0.05).

Respiratory activity from the spinal C2 ventral root was recorded using an ACSF-filled glass electrode (100 kOmega ), amplified ×70,000, band-pass filtered (100 Hz-3 kHz), digitized at 20 kHz, and written to computer disk.

Rectified and integrated C2 activity (tau  approx 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.


    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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Table 1. Changes in respiratory period as a function of inflation delay



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Fig. 2. A: transient inflation at 50 ms delay increases respiratory frequency. C2, rectified integrated ventral root signal. Gray bar, phasic inflation. B: effect of transient inflation attenuates with delay from inspiratory onset. Columns, means of bout means; error bars, adjusted mean squared error of all bout means; dots, individual bout means (n = 10). Inflation at delays of 50-1500 ms produced respiratory periods significantly shorter than control (asterisks, P < 0.05). All test means other than 500 and 1000 ms delay are significantly different from each other (P < 0.05).

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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Fig. 3. Increased respiratory frequency occurs without inspiratory shortening. A: pooled mean of control (solid line), 50 ms delay inflation (gray area), and 1000 ms delay inflation (dashed line) inspiratory burst envelopes (n = 5) normalized to a maximal amplitude of 1. B: both test conditions significantly shortened the respiratory period (diagonal bars, P < 0.05), but only 50 ms delay inflation significantly shortened inspiratory burst duration (white bars, P < 0.05).


    DISCUSSION
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INTRODUCTION
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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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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