Morphologic Changes in the Airways Induced by Recurrent Exposure of Acetylcholine in the Guinea Pig1- 3
ATSUSHI NAGAI, HIDETAKA INANO, and TAKAO TAKIZAWA Introduction
It is wellknown that airway smooth muscle is increased in patients with bronchial asthma (1-8). However, the relationship between increased muscle and incidence of asthma remains uncertain since previous studies have used materials obtained from autopsy cases. Whether recurrent bronchoconstriction results in increased airway muscle, and if so the location of the increased muscle and its effect on airway dimension, has not been elucidated. We have therefore examined the amount of smooth muscle and the morphologic changes in the airways specifically associated with recurrent asthmalike breathing of guinea pigs induced by acetylcholine exposure. Methods Thirteen male Hartley guinea pigs 4 wk of age were used in this study. The experimental animals wereexposed to 5,000 y of acetylcholine (Ach) mist (distilled with saline solution) in a chamber 40 x 30 x 35 ern, These animals demonstrated asthmalike breathing within 3 to 5 min of exposure (asthmalike breathing after the exposure to Ach occurred sooner with repeated exposures), and such breathing was maintained for about 15 min in the chamber until near asphyxia. After opening the chamber to allow breathing to return to normal, the animals were again exposed to Ach for at least 150min approximately 10exposures a day for 7 days a weeks for 2 or for 3 wk (table 1).Control animals were exposed to saline mist and treated in the same manner as the experimental animals except for exposure to Ach. After the 2 or the 3 wk of exposure and after measurements of body weight, the animals were injected intraperitoneally with an overdose of sodium pentobarbital, and the lungs were quickly excised. This approach minimized the postmortem bronchoconstriction seen in guinea pig lungs (9). Subsequently, the lungs were inflated for at least 24 h with 2.5010 glutaraldehyde (cacodylate buffer) through an intratracheally cannulated tube at a constant pressure of 25 em H 2 O. Fixed lung volume was determined by the water displacement method of Scherle (10). Tissue sections 5 urn thick were taken from the midsagittal slice and stained with Masson trichrome. 172
SUMMARY The morphologic changes in peripheral airways of guinea pigs derived from asthmalike breathing induced by acetylcholine (Ach) exposure were studied. In the Ach·exposed animals, the smooth muscle in the respiratory units (alveolar mouth of the alveolar duct) as well as in the peripheral airways was increased. The internal and external diameters of the peripheral airways were not significantly different between Ach·exposed and control animals. In addition, the Ach-exposed animals had higher specific diaphragm weights (diaphragm to body weight). Weconclude that recurrent asthmalike breathing brings about increased smooth muscle, but the increased muscle contrIbutes little to airway dimensions. We speculate that an increased airway wall thickness inside the outermost layer of smooth muscle may playa role in increasing airway reactivity. AM REV RESPIR DIS 1990; 142:172-176
Morphometry The following measurements and calculations were made in order to assess the amount of airway smooth muscle and airway dimensions in the membranous bronchioles (nonalveolated, noncartilagenous airways): the volume proportion of airway smooth muscle (the ratio of the area of smooth muscle to the area of airway wall) (11), the ratio of smooth muscle thickness to airway wall thickness (Rmus) (figure 1), and the internal and external adventitial diameters of the airways. In addition, in the 3-wk experimental animals, we measured airway wall thickness according to the method of Moreno and coworkers (12). This measurement is based on the concept that increased airway wall thickness is associated with increased airway reactivity (13). The airway wall thickness was expressed as the proportion of the total area formed by a circle enclosing the outermost layer of airwaysmooth muscle occupied by the wallcrosssectional area. These measurements were made by projecting images onto a computerassisted graphics tablet. Because the muscle in the alveolar mouth of the alveolar duct (these distal muscular bands appear as "knobs" in cross section) (14) was found to be prominent in light microscopic observation (15), the amount of muscle in the region was assessed by intuitively assigning the amounts to three groups. A large amount was identified as obvious mass structure, and a small amount was identified as very scanty muscular structure. The intermediate amount was approximately halfway between these two. Any alveolar process in the top (alveolar mouth) without muscle was not included in the assessment since the alveolar mouth can be missed by oblique cutting. The assessments were made at stratified random fields (n = 5) in each animal. One field included about six areas of the appropriate
alveolar process to be assessed. The muscularity was represented as a percentage ratio of the number of each grading in the alveolar process to the total number of alveolar processes. Additionally, the diaphragms of the 3-wk experimental animals were excised and weighed. The two groups of animals, Ach-exposed and control, were compared using Student's t test and the random-effects regression method of Feldman (16). Ridit analysis was applied to compare the amount of muscle in the terminal respiratory units (the amount of muscle of alveolar mouth in the alveolar duct). Probability values less than 0.5 were considered significant.
Results
There was no difference in specific lung volume (lung volume/body weight) between Ach-exposed (0.032 ± 0.004, mean ± SE) and control (0.032 ± 0.002) animals. Diaphragm weights, expressed as a percentage of body weight, were significantly (p < 0.05) increased in the 3-wk Ach-exposed animals (0.367 ± 0.016) compared with those in the control animals (0.315 ± 0.010).
(Received in original form July 7, 1989 and in revised form January 2, 1990) 1 From the Department of Medicine 1, Tokyo Women's Medical College, Tokyo, Japan. 2 Supported by Grant-in-Aid for Scientific Research No. 61570397. 3 Correspondence and requests for reprints should be addressed to Takao Takizawa, M.D., Department of Medicine 1, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan.
173
EFFECTS OF ACETYLCHOLINE EXPOSURE ON THE GUINEA PIG
Groups Ach-exposed animals
TABLE 1
Light Microscopic Findings
STUDY DESIGN
In the Ach-exposed animals, smooth muscle was prominent in the alveolar mouths of alveolar ducts as well as in the membranous bronchioles (figure 2). In contrast, the smooth muscle in the larger cartilagenous airways was similar in distribution in the two groups of animals. There was no obvious difference in alveolar structure, cellular infiltration, and basal lamina of bronchioles between the two groups.
Exposure Period' (wk)
Animals (n)
2
3 4
3 Control animals
2
3
3 3
• Animals were exposed for 7 days/wk.
Morphometry
b
smoo t h muscle
Fig. 1. Measurements of the thickness ratio of smooth muscle to airway wall (Rmus). The measurements are made on the line of maximal diameter in the minor axis . The basement membrane was used in the measurements of airway wall th ickness and internal diameter (a). b = external diameter. Rmus = (c + c')/(b - a).
Because there were no morphologic differences in the larger cartilagenous airways, quantitative assessments were performed only in the peripheral airways. The correlation between two assessments such as the volume proportion of smooth muscle and the ratio of muscle thickness to the airway wall (Rmus) is shown in figure 3. It will be noted that there was a significant correlation between them (r = 0.48, p < 0.05). The ratio of muscle thickness (Rmus) to internal diameter ofmembranous bronchioles is shown in figure
4. As is clear, the ratio of muscle thickness was increased with decreasing internal diameter of smaller airways in the two groups (Ach-exposed and control ani mals). Also in the Ach-exposed animals, the muscle thickness was greater than that in the control animals, and the difference was significant (p < 0.05). The amounts of smooth muscle in the Achexposed and the control animals are compared in figure 5. Airway smooth muscle, as assessed by both volume proportion (figure 5A) and thickness ratio (figure 5B), was significantly increased in the animals exposed to Ach for 2 wk as well as for 3 wk when compared with that in control animals. The airway wall thickness (PW), as defined by Moreno and coworkers (12) using tangentially cut bronchioles, was measured. As shown in figure 6, the Ach-exposed animals had a higher PW value in the various sized airways, and the mean PW (0.268) in the Ach-exposed animals (3-wk exposure) was increased compared with that (0.236) in the control animals. The results of semiquantitative assess-
. ,. Fig. 2. The smooth muscle layer of the bronchiole is prominent in the Ach-exposed animal (right) compared with that in the control animal (left) (original magnification: x300).
174
NAGAI, INANO, AND TAKIZAWA
100
A
:J
E
•
0
a:
Fig. 3. There is a significant correlation betwen the volume proportion of smooth muscle (Vvmus) and Rmus. Open circles control; closed circles = 3w-Exp.
8" • o •
0
~ 30
E
00
8
r P
ATSUSHI NAGAI, HIDETAKA INANO, and TAKAO TAKIZAWA Introduction
It is wellknown that airway smooth muscle is increased in patients with bronchial asthma (1-8). However, the relationship between increased muscle and incidence of asthma remains uncertain since previous studies have used materials obtained from autopsy cases. Whether recurrent bronchoconstriction results in increased airway muscle, and if so the location of the increased muscle and its effect on airway dimension, has not been elucidated. We have therefore examined the amount of smooth muscle and the morphologic changes in the airways specifically associated with recurrent asthmalike breathing of guinea pigs induced by acetylcholine exposure. Methods Thirteen male Hartley guinea pigs 4 wk of age were used in this study. The experimental animals wereexposed to 5,000 y of acetylcholine (Ach) mist (distilled with saline solution) in a chamber 40 x 30 x 35 ern, These animals demonstrated asthmalike breathing within 3 to 5 min of exposure (asthmalike breathing after the exposure to Ach occurred sooner with repeated exposures), and such breathing was maintained for about 15 min in the chamber until near asphyxia. After opening the chamber to allow breathing to return to normal, the animals were again exposed to Ach for at least 150min approximately 10exposures a day for 7 days a weeks for 2 or for 3 wk (table 1).Control animals were exposed to saline mist and treated in the same manner as the experimental animals except for exposure to Ach. After the 2 or the 3 wk of exposure and after measurements of body weight, the animals were injected intraperitoneally with an overdose of sodium pentobarbital, and the lungs were quickly excised. This approach minimized the postmortem bronchoconstriction seen in guinea pig lungs (9). Subsequently, the lungs were inflated for at least 24 h with 2.5010 glutaraldehyde (cacodylate buffer) through an intratracheally cannulated tube at a constant pressure of 25 em H 2 O. Fixed lung volume was determined by the water displacement method of Scherle (10). Tissue sections 5 urn thick were taken from the midsagittal slice and stained with Masson trichrome. 172
SUMMARY The morphologic changes in peripheral airways of guinea pigs derived from asthmalike breathing induced by acetylcholine (Ach) exposure were studied. In the Ach·exposed animals, the smooth muscle in the respiratory units (alveolar mouth of the alveolar duct) as well as in the peripheral airways was increased. The internal and external diameters of the peripheral airways were not significantly different between Ach·exposed and control animals. In addition, the Ach-exposed animals had higher specific diaphragm weights (diaphragm to body weight). Weconclude that recurrent asthmalike breathing brings about increased smooth muscle, but the increased muscle contrIbutes little to airway dimensions. We speculate that an increased airway wall thickness inside the outermost layer of smooth muscle may playa role in increasing airway reactivity. AM REV RESPIR DIS 1990; 142:172-176
Morphometry The following measurements and calculations were made in order to assess the amount of airway smooth muscle and airway dimensions in the membranous bronchioles (nonalveolated, noncartilagenous airways): the volume proportion of airway smooth muscle (the ratio of the area of smooth muscle to the area of airway wall) (11), the ratio of smooth muscle thickness to airway wall thickness (Rmus) (figure 1), and the internal and external adventitial diameters of the airways. In addition, in the 3-wk experimental animals, we measured airway wall thickness according to the method of Moreno and coworkers (12). This measurement is based on the concept that increased airway wall thickness is associated with increased airway reactivity (13). The airway wall thickness was expressed as the proportion of the total area formed by a circle enclosing the outermost layer of airwaysmooth muscle occupied by the wallcrosssectional area. These measurements were made by projecting images onto a computerassisted graphics tablet. Because the muscle in the alveolar mouth of the alveolar duct (these distal muscular bands appear as "knobs" in cross section) (14) was found to be prominent in light microscopic observation (15), the amount of muscle in the region was assessed by intuitively assigning the amounts to three groups. A large amount was identified as obvious mass structure, and a small amount was identified as very scanty muscular structure. The intermediate amount was approximately halfway between these two. Any alveolar process in the top (alveolar mouth) without muscle was not included in the assessment since the alveolar mouth can be missed by oblique cutting. The assessments were made at stratified random fields (n = 5) in each animal. One field included about six areas of the appropriate
alveolar process to be assessed. The muscularity was represented as a percentage ratio of the number of each grading in the alveolar process to the total number of alveolar processes. Additionally, the diaphragms of the 3-wk experimental animals were excised and weighed. The two groups of animals, Ach-exposed and control, were compared using Student's t test and the random-effects regression method of Feldman (16). Ridit analysis was applied to compare the amount of muscle in the terminal respiratory units (the amount of muscle of alveolar mouth in the alveolar duct). Probability values less than 0.5 were considered significant.
Results
There was no difference in specific lung volume (lung volume/body weight) between Ach-exposed (0.032 ± 0.004, mean ± SE) and control (0.032 ± 0.002) animals. Diaphragm weights, expressed as a percentage of body weight, were significantly (p < 0.05) increased in the 3-wk Ach-exposed animals (0.367 ± 0.016) compared with those in the control animals (0.315 ± 0.010).
(Received in original form July 7, 1989 and in revised form January 2, 1990) 1 From the Department of Medicine 1, Tokyo Women's Medical College, Tokyo, Japan. 2 Supported by Grant-in-Aid for Scientific Research No. 61570397. 3 Correspondence and requests for reprints should be addressed to Takao Takizawa, M.D., Department of Medicine 1, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan.
173
EFFECTS OF ACETYLCHOLINE EXPOSURE ON THE GUINEA PIG
Groups Ach-exposed animals
TABLE 1
Light Microscopic Findings
STUDY DESIGN
In the Ach-exposed animals, smooth muscle was prominent in the alveolar mouths of alveolar ducts as well as in the membranous bronchioles (figure 2). In contrast, the smooth muscle in the larger cartilagenous airways was similar in distribution in the two groups of animals. There was no obvious difference in alveolar structure, cellular infiltration, and basal lamina of bronchioles between the two groups.
Exposure Period' (wk)
Animals (n)
2
3 4
3 Control animals
2
3
3 3
• Animals were exposed for 7 days/wk.
Morphometry
b
smoo t h muscle
Fig. 1. Measurements of the thickness ratio of smooth muscle to airway wall (Rmus). The measurements are made on the line of maximal diameter in the minor axis . The basement membrane was used in the measurements of airway wall th ickness and internal diameter (a). b = external diameter. Rmus = (c + c')/(b - a).
Because there were no morphologic differences in the larger cartilagenous airways, quantitative assessments were performed only in the peripheral airways. The correlation between two assessments such as the volume proportion of smooth muscle and the ratio of muscle thickness to the airway wall (Rmus) is shown in figure 3. It will be noted that there was a significant correlation between them (r = 0.48, p < 0.05). The ratio of muscle thickness (Rmus) to internal diameter ofmembranous bronchioles is shown in figure
4. As is clear, the ratio of muscle thickness was increased with decreasing internal diameter of smaller airways in the two groups (Ach-exposed and control ani mals). Also in the Ach-exposed animals, the muscle thickness was greater than that in the control animals, and the difference was significant (p < 0.05). The amounts of smooth muscle in the Achexposed and the control animals are compared in figure 5. Airway smooth muscle, as assessed by both volume proportion (figure 5A) and thickness ratio (figure 5B), was significantly increased in the animals exposed to Ach for 2 wk as well as for 3 wk when compared with that in control animals. The airway wall thickness (PW), as defined by Moreno and coworkers (12) using tangentially cut bronchioles, was measured. As shown in figure 6, the Ach-exposed animals had a higher PW value in the various sized airways, and the mean PW (0.268) in the Ach-exposed animals (3-wk exposure) was increased compared with that (0.236) in the control animals. The results of semiquantitative assess-
. ,. Fig. 2. The smooth muscle layer of the bronchiole is prominent in the Ach-exposed animal (right) compared with that in the control animal (left) (original magnification: x300).
174
NAGAI, INANO, AND TAKIZAWA
100
A
:J
E
•
0
a:
Fig. 3. There is a significant correlation betwen the volume proportion of smooth muscle (Vvmus) and Rmus. Open circles control; closed circles = 3w-Exp.
8" • o •
0
~ 30
E
00
8
r P
