nervously-mediated changes in tracheal volume on medullary stimulation of dogs

Respiration Physiology (1970) 9, 348-355; North-Holland Publishing Company, Amsterdam

NERVOUSLY-MEDIATED CHANGES IN TRACHEAL VOLUME ON

MEDULLARY STIMULATION OF DOGS

5. F. STEIN AND if. G. WIDDICOMBE

University Laboratory of Physiology, Oxford, England

Abstract. Changes in the volume of an isolated innervated segment of trachea, minute volume, heart rate and blood pressure have been measured during stimulation of the medullary “respiratory centres” of anaesthetised dogs. Changes in ventilation were nearly always accompanied by changes in tracheal volume. The changes in tracheal volume were not solely the reflex effect of the changes in ventilation, since they occurred in paralysed animals artificially ventiIated at tixed levels. Paralysis often reversed, but never eliminated, the tracheal response to stimulation at a particular site. Tracheal responses were highly dependent on the frequency of stimulation, high frequencies tending to lead to dilatation, low to constriction. This frequency-dependent effect was observed less commonly with the respiratory response and rarely with the cardiovascular response. ‘I here were no correlations between sites of stimulation and respiratory, tracheal or cardiovascular responses.

Respiratory centers Tracheal motivity Respiratory reflexes Tracheal volume

Vagal motoneurones efferent to tracheobronchial smooth muscle, and presumably

constrictor in function, usually have a respiratory rhythm; their average discharge

is increased in several conditions which stimulate breathing, such as hypoxia, hyper-

capnia and deflation of the lungs, and is decreased when breathing is inhibited by

the Hering-Breuer inflation and the carotid sinus baroreceptor reflexes (VINOGRADOVA,

1955; WIDDICOMBE, 1961,1966). It is therefore reasonable to suppose that the neurones

have functional links in the medulla with the respiratory centres.

This paper describes an attempt to see if there is a correlation between the respira-

tory and the tracheal muscular responses to electrical stimulation of the medullary

reticular formation of anaesthetised dogs. Airway responses (assessed by indirect

methods) to stimulation of the cerebral cortex have been described (FRANCOIS-

FRANK, 1888); however, there seem to be no corresponding experiments on the

brainstem, or any experiments with unequivocal methods of determining changes in

airway calibre.

Accepted for publication 15 December 1969.

348

MEDULLARY CONTROL OF TRACHEA 349

Methods

Nine unselected mongrel dogs were anaesthetised with either pentobarbitone sodium

(Nembutal, Abbott; 30 mg/kg i.v.) or chloralose and urethane (2 ml/kg of a mixture

of 2.5 g/100 ml chloralose and 25 g/l00 ml urethane, iv.). All animals were paralysed

at some time in the experiment with the short acting muscle relaxant succinylcholine

(0.05 mg/kg). A femoral artery and vein were cannulated, the former for recording

blood pressure with a capacitance manometer (Southern Instruments), the latter for

administering drugs.

Tracheal volume changes were measured by the method of NADEL and WIDDICOMBE

(1962) of which the following is an abbreviated description. Two T-shaped brass

cannulae were tied into the upper and lower ends of the cervical trachea, care being

taken to avoid damage to the recurrent laryngeal nerves. The upper cannula blocked

the cephalic and the lower blocked the caudal end of the segment of trachea isolated

in this way. The lower cannula allowed ventilation of the lungs through the thoracic

trachea. The cannulae were rigidly clamped to the table so that no tracheal volume

change could be caused by respiratory or other movements. Volume changes in the

segment were recorded through the upper cannula with a microspirometer actuating

a capacitance transducer.

Ventilation was monitored through the lower cannula with a pneumotachograph

(Fleisch head and Godart Inductance manometer), and a volume record obtained

by electrical integration of signal (Godart, GM 0577). Tntrapleural pressure was

recorded from a lower right intrapleural space through a Malecot catheter, using a

capacitance manometer (Southern Instruments). Arterial blood pressure, tracheal

segment volume change, tidal volume and intrapleural pressure were displayed on

an oscilloscope (Tektronix 551) and photographed on 7 cm paper. A stimulus artefact,

indicating stimulus timing, duration and intensity, was superimposed on the tracheal

volume record.

After these preparations the animal was mounted prone in a stereotaxic holder

with head and vertebral clamps, and the medulla was exposed. The cerebellum was

left intact. The electrodes were mounted normal to the surface of the medulla and

their lateral and anteroposterior positions were defined by coordinates centred on the

obex, whilst their depth was measured from the surface of the medulla. The position

of the electrode tip was chosen so as to avoid cranial nerve nuclei. In the first 4 dogs

tungsten steel needles coated with a thin layer of varnish (tip diameter approximately

40 p, DC resistance 10 kohms) were used. Later, glass microelectrodes with silver

wile (tip diameter 10 p, 500 kohms resistance) were used. The current passing during

stimulation was measured from the voltage drop across a known resistance.

A general purpose stimulator (KAY, PHILLIPS and TEAL, 1958) which gave a

frequency range 0 to 300 Hz, voltage 0 to 60 V, stimulus duration 0.25 to 10 msec,

isolated from earth, was used. This paper only reports qualitative results and, although

quantitative variations in response resulted from altering other stimulus parameters,

only in the case of stimulus frequency were consistent qualitative differences observed.

350 J. F. STEIN AND J. G. WIDDICOMBE

Routinely, therefore, in control trials stimulus duration, amplitude and train duration

were standardised for each animal, such that the tracheal response from an active

point approximated to the maximal reflex constrictions and dilatations previously

obtained by tracheal occlusion and large lung inflations respectively in the same

animal. In the first experiments, stimulus frequency was kept in the range IO-40

shock per second on the assumption that this was the likely firing frequency of the

neurones involved. However, it soon became clear that, even if all other parameters

were kept constant, increasing the frequency of the stimulus would often reverse the

direction of the tracheal response. So later each spot was routinely tested at both

“high” (30-50 shocks/set) and “low” frequencies (IO-30 shocks/set).

v TR B.P.

A

B

C

C -w&-j .*......_.._..........

\I CL

C[f

D 3

.a..................

11 [[-

Fig. 1. Traces in each panel from above downwards: tracheal volume (VTR) with stimulus artefact superimposed (constriction downwards); femoral artery blood pressure (B.P.); 1 second time marker, with event signal; tidal volume (VT) (zero reset in panel A was necessitated by integrator drift); transpulmonary pressure (PTP). All panels show stimulation at a point 2 mm rostrally and 3 mm lateral from the obex and 4 mm below the surface of the medulla at a stimulus intensity of 10 /cA. A. Spontaneous breathing. Low frequency stimulation (.20 Hz) causes tracheal constriction, hyperpnoea, hypertension and bradycardia. B. Spontaneous breathing. High frequency stimulation (SO Hz) causes tracheal dilatation, hypopnoea, hypertension and bradycardia. C. Paralysed, artificially ventilated. Low frequency stimulation (20 Hz) causes tracheal constriction hypertension and bradycardia. D. Paralysed, artificially ventilated. High frequency stimulation (50 Hz) causes tracheal dilatation,

hypertension and bradycardia.


Results

Fig. 1 shows typical tracheal and ventilatory responses to stimulation. The tracheal

response was generally either a dilatation or a constriction. The time-courses of

dilatations and constrictions were very similar with latencies of approximately 5 sec.

The ventilatory response to stimulation was often complex. Sometimes a tachypnoea

merged into apnoea and gave way to tachypnoea again; sometimes bradypnoea

slowed to a standstill and then bradypnoea broke through again. Tidal volume

usually decreased with tachypnoea and increased with bradypnoea. We therefore

classified the overall changes in ventilation qualitatively according to whether the

minute volume over the full length of the stimulus, compared with that over a similar

control period, increased or decreased.

Advancing the electrode by no more than 0.5 mm often reversed the tracheal and/or

ventilatory responses to identical stimuli. Also increasing the frequency of stimulation

often reversed the responses from the same point. High frequencies characteristically

gave rise to tracheal dilatation and hyperpnoea, and their use led to the observation

of frequency-dependent reversal of responses (fig. 1).

TRACHEAL AND VENTILATORY RESPONSES IN SPONTANEOUSLY-BREATHING ANIMALS

Since we intended to correlate tracheal and breathing responses to stimulation we

have excluded all those medullary points whose stimulation failed to elicit a breathing

response at all. All the remaining “respiratory centre” points gave rise to a tracheal

response at one or other of the stimulus frequencies.

High and low frequency stimulations were carried out at each of 69 spots. High

frequency stimulations caused tracheal dilatation at 45 points, and low frequency

stimulations caused constriction at 62 points. Forty-three (65 %) points showed the

frequency-dependent reversal of tracheal response. At all but two of the 26 points

not showing frequency-dependent reversal of the tracheal response, both high and

low frequency stimulation caused tracheal constriction.

The dominant ventilatory response was an increase in minute volume at either

frequency of stimulation (66% of all tests). High stimulus frequencies caused hyper-

pnoea in 78 % of tests, whereas low frequencies caused hyperpnoea and hypopnoea

about equally. The frequency dependent effect (high frequencies causing hyperpnoea

and low frequencies hypopnoea usually) was demonstrated at 17 points (25x), and

was thus not so common with the ventilatory as with the tracheal responses (65%).

CORRELATION BETWEEN TRACHEAL AND VENTILATORY RESPONSES

Stimulation of 33 % of medullary points in spontaneously-breathing animals (usually

at low frequency) caused hyperpnoea with tracheal constriction. 30% led to hyper-

pnoea and tracheal dilatation. 25 % led to hypoventilation and tracheal constriction,

whilst the rarest response was hypoventilation with tracheal dilatation (3%). In the

remaining 9% of tests either ventilation or tracheal volume failed to change at one

or other of the stimulus frequencies.

There are several possible explanations of these associations. Activation of


the Hering-Breuer inflation reflex by increased lung volume dilates the airways

(LOOFBOURROW, WOOD and BAIRD, 1957; WIDDICOMBE and NADEL, 1963). Asphyxial

stimuli cause nervously-mediated bronchoconstriction (NADEL and WIDDICOMBE,

1962). Therefore tracheal volume might have been influenced reflexly by changes in

ventilation in response to medullary stimulation. An alternative explanation is that

the medullary neuronal systems have direct efferent control over airway tone in-

dependent of ventilatory reflexes.

EXPERIMENTS WITH PARALYSED ANIMALS

To see if the ventilatory response to medullary stimulation determines the tracheal

response, we carried out a series of experiments with paralysed animals. Ventilatory

responses to stimulation were thus eliminated, and tracheal volume changes were

probably subject only to direct medullary control. All 16 out of 16 points in the

medulla stimulated only when the animal was paralysed showed changes in tracheal

volume, so the tracheal responses were not solely due to the ventilatory responses.

For 15 of the points the frequency-dependent effect could be elicited by choosing

the control conditions carefully. Increasing or decreasing the minute volume of the

ventilation pump dilated or constricted the trachea respectively, presumably by lung

stretch reflexes and nervously-mediated blood gas actions. At a fixed medullary spot

high frequency stimulations were more likely to yield tracheal dilatation if control

trachea constriction had been first induced by reducing pump minute volume, and

low frequencies were more likely to yield constriction if control tracheal dilatation

had been first induced by increasing pump minute volume. In this way 15 of the

16 points were induced to show the frequency-dependent effect.

POINTS STIMULATED BEFORE AND AFTER PARALYSIS

We tried to determine whether elimination of the ventilatory response to medullary

stimulations would alter the pattern of the tracheal response to stimulation at the

same point. Identical series of high and low frequency stimulations were made at

nine points before and after the animal had been paralysed with succinylcholine and

artificially ventilated at a level that permitted either tracheal dilatation or constriction

(i.e. a total of 36 stimulations). As before, paralysis never entirely eliminated the

tracheal responses present whilst the animal was breathing spontaneously; however

for 8 of the 18 stimulations before paralysis, repetition after paralysis caused a

reversal of the response with the same stimulus parameters. This reversal suggested

that in the spontaneously breathing animal a direct tracheal response to medullary

stimulation could be overwhelmed by secondary bronchomotor effects of the simul-

taneous ventilatory response. For four of the eight stimulations where paralysis

reversed the direction of response, stimulation had caused bronchoconstriction with

strong inhibition of breathing when the animal was breathing spontaneously. In the

remaining four instances bronchodilatation with increased breathing was converted

by paralysis to bronchoconstriction.

Seven points showed a frequency-dependent reversal of response when the animal

was breathing spontaneously (high frequency stimulation causing dilatation, low


frequency stimulation causing constriction). Paralysis eliminated the frequency-

dependence of some of these points but introduced others so that a different 7 points

showed the effect after paralysis. Two points showed the same tracheal response to

both high and low frequency stimulation before and after paralysis.

CARDIOVASCULAR RESPONSES TO MEDULLARY STIMULATION

Cardiovascular responses to the stimulations were frequent. They were noted in

some tests on 77 of the grand total of 94 points studied. For stimulations at the

responsive points blood pressure rose in 67% and fell in 33 ‘4. Heart rate rose in

24 % and fell in 76 %. No consistent frequency-dependence was seen. Only twice did

the blood pressure response reverse with changing frequency, a hypotensive effect

with low frequency stimulation changing to a hypertensive effect with high frequency

stimulation in both instances. For heart rate on one occasion a bradycardia was

converted to a tachycardia by increasing the frequency of stimulation. There was

no correlation between the cardiovascular responses and either airway or ventilatory

responses, which confirms the results of BACH (1952) with cats.

ANATOMICAL LOCALISATION OF POINTS

All the points were localised in relation to the obex by stereotaxic coordinates. They

lay within coordinates 3 mm caudal, 5 mm rostrally, between 2-4 mm laterally on

either side and 3-6 mm deep. We confirmed that the electrode tracks did not involve

cranial nerve nuclei, particularly n. tr. solitarius, n. vag. dorsalis or n. ambiguus by

examination of hand-cut slices of the medulla fixed with formalin in situ, under a

dissecting microscope. The electrode tracks were clearly visible; the responsive points

were in the medial reticular formation including the n. gigantocellularis. More

detailed histological control was judged unnecessary, as the size of the electrodes

and the probable amount of current spread rendered finer localisation of the electrode

tip irrelevant. With our classification there was never any spatial grouping of points

showing similar responses to stimulation, as might have been expected on the hypo-

thesis of anatomically-defined inspiratory or expiratory or airway control “centres”,

although as we have seen the airway responses were elicited from the same points

as the respiratory responses.

Discussion

BROOKHART (1940) has shown that stimulation of the medulla of dogs does not

produce as clear inspiratory and expiratory responses as in the cat, and that the two

“centres” are not anatomically distinct. The “respiratory” centres have been neither

so precisely localised nor so frequently studied in the dog compared with the cat.

The respiratory responses we obtained were similar to those of BROOKHART and we

confined our stimulations to the area shown by him to contain neurone systems

influencing breathing on stimulation. The anatomical correlation between points

giving respiratory and tracheal responses was consistent with the observations of

VINOGRADOVA (1955) and WIDDICOMBE (1961, 1966) on vagal efferent neurones. Vagal


tracheobronchial constrictor neurones in cats and dogs usually have a respiratory

rhythm and both their mean rate of firing and minute volume are changed in a

variety of physiological conditions.

The primary tracheal response could be complicated by secondary effects. The

hyperpnoea would have a dilator and hypopnoea a constrictor influence on the trachea

by lung reflexes (WIDDICOMBE and NADEL, 1963), and by blood gas changes (NADEL

and WIDDICOMBE, 1962). However, the experiments with paralysed dogs show that,

although these secondary effects may have modified the response in some instances,

there was always an underlying primary response. Similarly the baroreceptor reflex

has a tracheodilator component (NADEL and WIDDICOMBE, 1962), and catecholamine

secretion could have a direct dilator effect on the trachea. However, the lack of

correlation between tracheal and cardiovascular changes suggest that these complica-

tions were not important in our experimental conditions.

The tracheal responses depended on stimulus frequency. Although this frequency-

dependence was always exhibited with constant stimulus strength and duration at

the same spot, it is not suggested that a single functional group of neurones exerted

opposite actions on the trachea as frequency of firing changed. A likely explanation

is that the choice of stimulation frequency alters the balance of influences of a mixed

population of neurones with different frequency characteristics.

If, therefore, we were stimulating neurones or fibres with a “direct” effect on the

trachea, the question arises as to their nature. Fibre pathways afferent to the medulla

seem unlikely. The pulmonary stretch (Hering-Breuer inflation reflex) pathway has

the lowest electrical stimulation threshold and highest natural frequency of firing in

the vagus nerve. Stimulation of the central end of one cut vagus nerve at high fre-

quencies leads to airway dilation (KAHN, 1907; LOOFBOURROW et al., 1957; WIDDI-

COMBE and NADEL, 1963), but the reflex response also includes expiratory apnoea,

a combination rarely seen in our experiments. In addition examination of cut sections

never showed vagal nuclei (such as the n. tr. solitarius) on the electrode track of

points causing tracheal responses.

Similarly baroreceptor high frequency discharge dilates the trachea and causes

hypotension (NADEL and WIDDICOMBE, 1962), but in our experiments tracheal

dilatation was not consistently associated with hypotension. A similar lack of correla-

tion between the tracheal constrictor responses to medullary stimulation and respira-

tory and cardiovascular changes makes the involvement of airway constrictor

afferent pathways, e.g. tracheal cough receptors (KENT, NADEL and WIDDICOMBE,

1962), lung irritant receptors (MILLS, SELLICK and WIDDICOMBE, 1969; SELLICK and

WIDDKOMBE, 1969) or “deflation” type J receptors (PAINTAL, 1969) unlikely. The

lack of correlation between cardiovascular and respiratory responses on medullary

stimulation in the dog confirms the work of BACH (1952) with cats.

It is also unlikely that we were directly stimulating only vagal efferent pathways

to the trachea as such stimulation would give rise to consistent (not frequency-

dependent) bronchoconstriction (and presumably bradycardia), and our coordinates

were chosen so as to avoid the motor nucleus of the cranial nerves. The dilator

MEDIJLLARY CONTROLOFTRACHEA 355

responses could be due to direct stimulation of efferent sympathetic pathways only

if they have a firing frequency many times higher than peripheral sympathetic fibres

to the lungs (WIDDICOMBE, 1966) and elsewhere (HILLARP, 1960); and if the sym-

pathetic dilator influence is many times stronger than indicated by the available

evidence (WIDDICOMBE, 1963; GREEN and WIDDICOMBE, 1966).

We therefore conclude that we were stimulating medullary systems of neurones

with a strong action on autonomic outflow to the trachea, but that the tracheal

responses may be modified by secondary nervously-mediated effects in non-paralysed

animals. Stimulation of the parts of the medulla corresponding to the “respiratory

centres” does not activate groups of neurones with uniform motor actions on the

trachea and respiratory muscles; it is more likely that mixed populations of neurones

are involved, which would explain the lack of correlation between respiratory and

tracheal responses, and the stimulus frequency-dependence of the tracheal responses.

References

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nervously-mediated changes in tracheal volume on medullary stimulation of dogs

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