Respiration Physiology (1976) 27, 241-251;

EFFECT OF ANTIGEN GUINEA

North-Holland Publishing Company, Amsterdam

CHALLENGE ON SENSITIZED PIG TRACHEA

J. F. SOUHRADA

and D. W. DICKEY

Pulmonary Function Laboratory, National Jewish Hospital and Research Center, Denver, Colo. 80206, U.S.A.

Abstract.

Isolated

tracheal

acute and chronic in addition smooth

a significant

state was observed

(AT,,,)

(ITP) were obtained (AA and CHA). response

increase

as the potentiated,

active tension

tional differences animals

asthma

to the classic Schultz-Dale

muscle),

maximum

preparations

experimental

(the immediate

phasic,

mechanical

with airway

muscle was seen. This active

of ITP and was evaluated

development

or the active state of airway

pigs and animals

in the tone of sensitized

smooth

activities

rate of tension

guinea

that after in uifro antigen challenge,

increase

of the active state of airway

and maximum

in the tonic response

from control

It was observed

smooth

(dT/dt).

by both

No apparent

func-

muscle between AA and CHA

were found.

Tracheas

isolated

from

by wet, dry and relative The repeated physiological

administration

with CHA

demonstrated

smooth

muscle

hypertrophy

as measured

of the tracheas. of antigen

into the experimental

bath

was ineffective

in inducing

any

changes.

The administration of trachea

animals weights

comparable

of histamine

to ITP from control

animals

to those seen in both AA and CHA Airway

smooth

Experimental

muscle asthma

induced

changes

of mechanical

activities

animals.

Histamine Mechanical

activities

of trachea

After the administration of an antigen to bronchial preparations isolated from both asthmatic animals and asthmatic patients, a significant increase in the airway smooth muscle tension has been observed (Mongar and Schild, 1962; Schild et al., 1951). It has been suggested furthermore that this tension increase is responsible for clinically observed bronchoconstriction. The decrease of bronchial diameter is thus responsible for an increase in airway resistance and other consequent changes of pulmonary functions, a fact which has been documented in many studies (Bouhuys, 1974; Gold et al., 1972; Permutt, 1971). Recently we have described rhythmical changes of the tracheal diameter of rabbits and guinea pigs, at both the in viuo and in vitro levels (Souhrada and Dickey, 1976). A phasic movement of the tracheobronchial tree has previously been suggested by several investigators (Macklin, 1929; Zanni, 1927). These activities, sometimes Accepred,for publication 9 April 1976. 241

242

J. F. SOUHRADA

AND D. W.

DICKEY

called ‘airway peristalsis’ (Widdicombe, 1963) are slight contractions with a frequency of 3-5 per minute, superimposed on the rhythmic changes of airway diameter induced by respiration. Subsequent experiments have demonstrated their relative independence of any nerve supply and have also established the inhibitory effect of atropin administration (Souhrada and Dickey, 1976). Recently Dunnill et al. ( 1969) speculated that the loss of ciliated bronchial mucosa and the accumulation of exudate in the bronchi of an asthmatic patient can result in increased peristaltic action of the airway smooth muscle which later results in its hypertrophy and hyperplasia (Hossain, 1973). Thus, in the present study the hypothesis of increased ‘peristaltic’ action of airway smooth muscle during asthma was experimentally tested. The effect of acute and chronic experimental asthma on the spontaneous activities of airway smooth muscle was investigated using an in vitro antigen challenge of tracheal preparations from acutely and chronically sensitized animals.

Method ACUTE

EXPERIMENTAL

ASTHMA

Essentially, the experimental protocol of Stein et al. (1961) was used. Ten male guinea pigs (Hartley strain) were injected intraperitoneally three times, in three alternating days, with 0.25 ml of fresh, undiluted egg white. Two weeks were allowed for sensitization. Then, two tracheal preparations from each animal were removed and tested. Six guinea pigs were injected with 0.25 ml of saline, and two weeks later they were utilized as controls.

CHRONIC

EXPERIMENTAL

ASTHMA

Thirty-five male guinea pigs (Hartley strain) weighing 200 to 300 grams were provided free access to food and water. At the beginning of the experiment 22 of them were injected intraperitoneally with fresh, undiluted egg white (0.25 ml) and the control group (n = 8) was similarly treated with the same amount of saline (0.9 % NaCl). Then, after a two-week period, sensitized, experimental animals (group I; n = 22) were placed in a plastic box (30 x 18 x 25 cm) in the top portion of which a standard nebulizer (USV Corp.) was attached. Every 15 seconds, for a period of 5 minutes, a diluted solution of egg white (one percent by volume in saline was sprayed into the box. A second experimental group (group II ; n = 5) was not previously sensitized by egg white injection but received a continuous exposure of the aerosolized, diluted egg white for a period of 3 minutes, also administered through a nebulizer. In both experimental groups the animals were exposed to the aerosolized antigen daily (5 days per week) for a total period of 8 weeks.

MECHANICAL ACTIVITY OF SENSITIZED AIRWAY SMOOTH MUSCLE

243

During exposures to the aerosolized antigen, some guinea pigs in both experimental groups usually developed difficulty with breathing, frequently accompanied by wheezing, typically observed immediately after antigen exposure. By 20 to 25 minutes after the antigen exposure, however, most of these symptoms had disappeared. ISOLATED TRACHEAL PREPARATION

Under light pentobarbital anesthesia (30 mg/kg b. wt) a segment of trachea (7 mm long) was quickly removed, cleaned and placed in a physiological salt solution (PSS) of the following composition (in mM/l): NaCl 117.5; KC1 5.37; MgSO, . 7 H,O 0.56; NaHCO, 15.41; NaH,PO, 1.12; CaCl, 1.61. The osmotic activity of the PSS was 290&5 mosm/kg. The temperature of PSS was maintained at 37.5kO.l “C. The segment of trachea was then cut into two pieces, so that each tracheal preparation was composed of 4 to 5 cartilage rings. In this manner two preparations from each animal could be analyzed. The tracheal segment was mounted on a special holder and immersed in 80 cc of a 37.5 “C PSS in a muscle chamber. Throughout the experiment the bath in the muscle chamber was aerated with a gas mixture of 95% oxygen and 5 % carbon dioxide. pH, P,, and Pcol of the experimental solution were thus maintained at relatively steady values of 7.38 f 0.02,28 f 2 mm Hg and 460 + 5 mm Hg, respectively. The preparation was attached via a gold jeweler’s chain to an isometric force transducer (Grass FT 0.03) which was connected to a micrometer and a displacement transducer (Hewlett-Packard 7DCDT). The segment of trachea was always oriented with the trachealis muscle positioned on one side. Further details of the method have been previously described (Souhrada and Dickey, 1975, 1976; Stephens and Kroeger, 1970). The initial length of the preparation (L,) was defined to be zero microns and to be the length at which no appreciable resting tension was recorded (Stein et al., 1961). Then, each preparation was set at a length of 40 microns. With this degree of stretch, the circular shape of the trachea was changed so that the tension developed by the trachealis muscle was parallel with the vertical axis and is reported in grams. Due to the isometric conditions, the length of the preparation was constant throughout the experiment. At the end of the incubation period (75 minutes) 0.5 ml of diluted (1 : 100) fresh egg white (in 0.9 % NaCl) was added into the experimental chamber. Maximum active tension (AT,,,) and maximum rate of tension development (dT/dt) are the parameters used to describe the spontaneous activities of the tracheal preparations (Buccino et al., 1967). Data are reported as means and standard errors of the mean and comparisons for statistical significance were made by means of Student’s t-tests. The t-test for paired observations was used for intra-group comparisons and the r-test for independent samples for inter-group comparisons. The maximum P value acceptable for reporting significant differences was 0.05.

244

J. F. SOUHRADA AND D. W. DICKEY

Rt?SUltS ACUTE

EXPERIMENTAL

ASTHMA

Table 1 demonstrates that acute experimental asthma did not significantly affect the growth rate of asthmatic animals as compared with controls. Both wet and dry weights of tracheal preparations isolated from experimental animals were not significantly different from controls. The total amount of tissue water of experimental tracheas was, however, significantly increased as compared with controls. 1

TABLE Morphological

characteristics

of tracheal

preparations

isolated

from

controls

and asthmatic

animals

(acute asthma) Body weight

Final

at start

body weight

(9)

(g)

Controls

202

(n = 6) Acute

asthma

190

* Shows a statistical Data

represent

significance

means

of resting

preparation (mg of dry wt/kg b. wt)

6.9

304

+0.4

23.5

5.5

* 1.2

*8

73.5

42.5 +3.9

+0.7

+0.3

16.3*

36.5 k2.3

+0.7

US. acute asthma).

+ SE.

tension

after antigen

tracheal

Dry wt

26.2

TABLE Changes

Rel. wt of

of H,O (mg)

f 1.8

at P < 0.05 (controls

Percent

Wet wt

*21

+I6

preparation

(mg)

326

+I8

(n = 10)

Tracheal

(RT) and maximum

administration

2

active tension

into the experimental

(AT,,,)

chamber

of isolated

tracheal

preparation

(0.5 ml of 1 % egg white)

_ Resting

tension

Active tension

Rate of tension development

(9) Pre A Controls

0.61

P

Post A

N.S.

0.64

+0.16

(n = 12) Acute asthma

0.71

+0.16 < 0.001

kO.15

(n = 20) Pre A

(gisec)

(g)

before antigen

0.41

4.79

‘0.45 kO.09

represent

P

Post A

N.S.

kO.15

kO.22 administration; Data

PreA

< 0.001

0.41

0.08

kO.19

kO.02

0.99 *0.11

Post A - after antigen means

PreA

0.05

P N.S.

Post A 0.04 +0.02

< 0.05

kO.01

0.09 kO.01

administration.

*SE.

Table 2 summarizes results obtained after in vitro antigen administration to isolated tracheas of controls and experimental animals. The administration of 0.5 ml of diluted egg white, 1 : 100, into the experimental chamber, did not affect either resting or active tensions of the tracheal preparations obtained from controls,

MECHANICAL ACTIVITY OF SENSITIZED AIRWAY SMOOTH MUSCLE

245

On the other hand, it was observed that identical administration of antigen into the chamber with tracheal preparations from experimental animals produced an almost immediate increase in resting tension (RT): the well known Schultz-Dale reaction (fig. 1). The maximum values of resting tension were achieved at 4.43 kO.36 minutes following the administration of antigen. As seen from fig. 1, the rise in resting tension

4. 2.2 Fig. I. The effect of antigen (0.5 ml, 1 : 100 egg white in saline) administration on the resting tension (RT) and the spontaneous mechanical activities of the representative tracheal preparations isolated from a control animal (I) and an animal with acute experimental asthma (II) A-D shows a continuous record of the crucial portion of one experiment.

(RT) was followed immediately by a potentiation of mechanical activities as measured by maximum active tension (AT,,,) and maximum rate of tension development (dT/dt); see table 2. After approximately one and a half hours the resting tension as well as the spontaneous activities returned toward baseline values.

CHRONIC EXPERIMENTAL ASTHMA

Table 3 shows that no significant differences were found between the growth rates of

246

J. F. SOUHRADA

AND D. W. DICKEY

TABLE Morphological

characteristics

of tracheal

3

preparation

isolated

(I and II) with chronic

from controls

and experimental

groups

asthma

Body weight

Final

Tracheal

at start

body weight

~~~~~-~~

(g)

(9)

preparation

Percent

Rel. wt of

~

of H,O

tracheal

Wet wt

Dry wt

(mg)

(mg)

preparation (mg of dry wtjkg ofb. wt.)

Controls

Group

I

Experimental

Group

11

(n = 5)

* Shows a statistical represent

49.5*

15.1*

+ I.5

(n = 16) 71.9*

35.8, + I.4

(n=44) 69.l*

+0.8 (n = IO) (n = IO)

at P < 0.05 (controls

31.5

+0.8

Jro.5

k2.6

+27

significance

means

(n=44)

+0.4 (n=44)

727

366 +I5

11.1*

39.4* & 1.3

*I8

+8

74. I

+0.6 (n = 16) (n = 16)

622

339

(n = 22)

8.7

33.7 k2.2

*I5

*I7

Experimental

Data

548

278

(n = 8)

+ I.2

41.7* +2.9

(n = IO)

vs. experimental).

k SE.

animals with experimental asthma (both experimental groups I and II) and controls. It can be seen, however, that the sizes of tracheal preparations as measured by wet, dry and relative weights are significantly increased (P < 0.05) in both experimental TABLE Changes

of resting

development

(dT/dt)

tension

(RT),

of the isolated

after administration

maximum tracheal

of the antigen Resting

4

active

tension

preparations,

(AT,,,)

isolated

into the experimental

and

chamber

rate

of tension

with chronic

asthma,

(0.1 cc of I “/‘, egg white)

Active tension

tension

maximum

from animals

Rate of tension development

(g)

(g) Pre A I.17

Controls (n = 16) Experimental

Post A

N.S.

1.23

+0.21 Group

I

(n = 44) Experimental

P

1.80

kO.20 < 0.001

kO.20 Group

(n = IO)

II

1.90

< 0.001

+0.20 Pre A

before antigen

PreA 0.30

4.20

0.20 kO.04

4.50

administration; represent

P

Post A

N.S.

kO.05

kO.20

kO.20

Data

(g/se@

0.09

< 0.001

0.44 +0.07

N.S.

kO.03 Post A means

0.35 kO.06

0.07

P N.S.

t0.01 0.03

0.17

0.02 kO.004

Post A 0.06 +0.01

< 0.001

+0.01

kO.05 after antigen &SE.

PreA

0.06 kO.01

N.S.

administration.

0.03 &0.004

MECHANICAL ACTIVITY OF SENSITIZED AIRWAY SMOOTH MUSCLE

247

groups (I and II) as compared with controls. On the other hand, in both experimental groups a lesser amount of tissue water was also found as compared with the control group (table 3). After administration of antigen (0.5 ml, 1 : 100 egg white in saline) into the experimental chamber, tracheal preparations from both experimental groups demonstrated a significant increase (P < 0.001) in resting tension (RT) (see tables 4 and 5). On the contrary, no increase in resting tension (RT) occurred in the control group after a second, identical administration of antigen. The maximum values of resting tension were reached 6-10 minutes after administration of antigen (table 5). TABLE 5 Changes of resting tension (RT) response after antigen administration experimental asthma

(post A) in acute and chronic

Time to maximal response in resting tension post A (min)

Duration of response (min)

Time to maximal active tension of S.C. post A (min)

Acute asthma (n = 20)

4.43 kO.36

After 60 minutes RT still 38 % above

39.5 * 4.9

Chronic asthma Experimental Group I (n = 44)

6.50 +0.-/o

f

58.9 5.0

23.8 + 2.5

P*

N.S.

< 0.05

< 0.05

Chronic asthma Experimental Group II

9.50 k2.20

88.0 f 18.0

41.3 kl3.0

* Shows a statistical significance between groups I and II. Data represent means +SE.

Similarly, spontaneous mechanical activities of tracheas isolated from controls were not affected by antigen administration. However, in experimental group I the administration of antigen induced a significant (P -=c0.001) increase in the amplitude of spontaneous mechanical activities (AT,,,) and the rate of tension development (dT/dt). Due to a large distribution among the data in experimental group II, administration of antigen failed to produce a statistically significant difference in either AT,,, or dT/dt. In a separate set of experiments, tracheal preparations isolated from asthmatic animals were repeatedly exposed to an antigen (diluted egg white) challenge. As can be seen in fig. 2, the repeated exposure of sensitized tracheas failed to produce any increase in the resting tension and did not change the pattern of spontaneous mechanical activity of the tracheal preparations.

248

J. F. SOUHRADA

AND D. W. DICKEY

2, I, Fig. 2. The effect of repeated activity

of a representative

mental

Group

I). A-D

exposure tracheal

of antigen preparation

shows a continuous

on the resting isolated record

tension

and the spontaneous

from an animal of the crucial

with chronic

portion

mechanical

asthma

(Experi-

of one experiment.

Discussion

Our data demonstrate that the administration of antigen, which is known to increase the resting tension of sensitized smooth muscle (Dale, 1913; Schild et al., 1951; Schultz, 1910) can also significantly increase the active state of airway smooth muscle as measured by spontaneous mechanical activities. This phenomenon was seen in both experimental models of asthma, acute and chronic. The administration of antigen significantly affected both maximum active tension (AT,,,) and maximum rate of tension development (dT/dt). Thus, in view of our findings it is possible that the increase of mechanical ‘peristaltic’ activity of the trachea and the bronchial tree is related to the general picture of airway hyperreactivity observed in asthma (Reed, 1974). In the lungs of young patients dying during status asthmaticus, it was found (Dunnill et al., 1969) that one of the outstanding pathological features is the presence of exudate in the bronchial tree and the loss of the ciliated bronchial mucosa. Dunnill et al. (1969) speculated that a result of cilia loss could be the failure to clean the exudate which would, in turn, provoke increased ‘peristaltic’ action of airway smooth muscle. Our experimental data will in part meet this hypothesis. Our data suggest that the well known airway smooth muscle hypertrophy and hyperplasia described in asthma (Dunnill et al., 1969; Hossain,‘l973) might result from increased ‘peristaltic’ activities of the airway smooth muscle of the asthmatic lung. Increased

MECHANICAL ACTIVITY OF SENSITIZED AIRWAY SMOOTH MUSCLE

249

m~hani~l activity (i.e. increase in ‘airway peristalsis’) together with transient, antigen induced increase of the tone of airway smooth muscle could be factors contributing to smooth muscle hypertrophy and hyperplasia. No attempt was made in the present study to evaluate the degree of smooth muscle hypertrophy and hyperplasia; nevertheless, our data demonstrate a significant increase in the wet, dry and relative weights of the tracheal preparations seen in animals with chronic experimental asthma. On the other hand, these types of changes were not seen in experiments with acute asthma. The majority of studies concerned with experimental asthma have been performed on once-sensitized animals (acute asthma), a situation which is quite different from the human pathophysiology of asthma. The present study tried to resolve this apparent weakness of the model of acute ex~rimenta1 asthma. In spite of the frequent bronchoconstrictive episodes induced by the repeated administration of antigen (a situation more closely resembling the condition of human asthma), no qualitative differences between the models of acute and chronic asthma were noted. This can partly be explained by the fact that in the case of chronic experimental asthma, bronch~onstrictive responses to inhaled antigen ap~rently decreased after about four weeks of the exposures. The dyspnea, coughing and general poor being of animals, frequently seen at the beginning of the experiment, had usually disappeared by this time. In the present study no attempt was made to affect mechanical activities of trachea through pharmacological intervention. We recently obtained data, however, 7 .l

Fig. 3. The effect of histamine (0.25 ml; 0.1 % histamine dihydrochloride) on the resting tension (RT) and the spontaneousmechanicalactivitiesof a representativetracheal preparation isolated from a control animal. A-D shows a continuous record of the crucial portion of one experiment.

J. F. SOUHRADA AND D. W. DICKEY

250

indicating that histamine administration into an experimental chamber containing a segment of non-sensitized trachea induces a response similar to that observed in sensitized preparations treated with antigen, i.e. the increase in spontaneous me~hani~l activities (fig. 3). Thus it is possible that the observed phenomenon of the potentiation of mechanical activities of the trachea is mediated through histamine or histamine-like substances released during antigen-antibody reaction. Histamine release during antigen-antibody reaction and activation of H, receptors seem to be important mechanisms inducing airway constriction, particularly in guinea pigs (Bouhuys, 1974; Mongar and Schild, 1962 ; Vane, 1971). Since the described preparation is anatomi~lly denervated, it can be assumed that the described findings are initiated on the local level, without direct neural control (Nadel, 1973). Bronchoconstriction, as seen during an asthmatic attack, is at present visualized as a sudden, sustained increase in airway smooth muscle tone (Bouhuys, 1974; Per-mutt, 1971). In vitro data, obtained from human bronchial segments isolated from asthmatic lungs, represent sufftcient evidence supporting this hypothesis (Schild et al., 1951). Nevertheless, from the present study it is apparent that the increase in the tone of the smooth muscle is probably not only a consequence of the antigen-antibody reaction in the lung. instead of seeing asthmatic airway obstruction as a static narrowing of airway diameter, we should visualize an asthmatic airway obstruction as a mechanical narrowing accompanied by superimposed phasic activities, i.e. increased ‘airway peristalsis.’ It will require further effor to define the significance of these findings for human asthma and other airways pathologies. Indeed, radiographic data shows that the movements of trachea and main bronchi were much more pronounced in a bronchitic lung than in a healthy human lung (Holden and Ardran, 1957).

Acknowledgment The authors would like to express their thanks to Mrs. Patty Dierker for her excellent clerical assistance.

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