initially we performed a pilot study with a yorkshire pig model , but because thin - section ct showed that the bronchi of this animal were relatively thick - walled and small - calibered , and the response to airway stimulating agents was thus minimally visible , the model was rejected as unsuitable .
these animals have relatively thin - walled and large - calibered bronchi , and ct thus demonstrates airway reactivity ; they are , in addition , relatively easy to handle .
eight mongrel dogs weighting 10 - 12 kg underwent fasting for 6 hours prior to anesthesia , and atropine ( daehan atropine ; daehan pharmaceutical , seoul ) was injected intramuscularly .
the dogs were anesthetized by intramuscular injection of 0.2 ml / kg of a mixture of 1ml of 2% xylazine hydrochloride ( rompun ; bayer korea , seoul , korea ) and 1ml of 10% ketamine hydrochloride ( ketara ; yuhan yanghang , seoul , korea ) .
anesthesia was maintained by intravenous injection of 5 - 7 mg / kg of thiopental sodium ( pentothal ; choong wae pharmacy , seoul , korea ) . immediately after the induction of anesthesia
, intubation was performed using a 7.5-mm inside - diameter endotracheal tube and a bear-2 adult volume ventilator ( bear medical systems corporation , riverside , cal . )
provided ventilation at a rate of 20 breaths/ minute and a tidal volume of 300 ml . to minimize motion artifacts and achieve constant lung volumes during ct scanning ,
the animals were paralyzed by intravenous injection of 1 mg/ kg of succinylcholine chloride ( quelin ; abbott laboratories , chicago , ill . ) at functional residual capacity ( 11 ) .
it has been reported that intravenous methacholine induces constriction only in the airways , whereas aerosolized methacholine causes constriction both in the airways and in the parenchyma ( 12 - 13 ) , and to induce bronchoconstriction , intravenous bolus injection of 0.1 mg / kg methacholine chloride ( provocholine ; roche , u.s.a . ) was therefore performed . to induce bronchodilation ,
2 ml of 1% salbutamol ( ventolin ; glaxowellcome ) was inhaled for 1 minute through a nebulizer connected to the ventilator . in our study , the procedure adopted by amirav et al .
( 15 ) was modified in such a way that initially , each dog underwent control thin - section ct , and after subsequent methacholine injection , the second series of ct scans was immediately obtained . after a 5-min delay ,
five minutes later , the final series of scans was obtained . in order to demonstrate the relationship between morphologic airway reactivity and physiologic parameters , peak airway pressure ( pap )
was measured through a pressure gauge connected to the ventilator immediately before ct scanning . during the early period
when spiral ct was not available , a ct / t 9800 quick scanner ( general electric medical system , milwaukee , wis . )
the number of slices was five to seven , each 3 cm below the tracheal carina and just above the diaphragm with an interval of 3-mm .
scanning parameters were 1.5-mm collimation , 1.5-mm interslice gap , 140 kvp , 170 ma and 1.5-sec scan time .
for the last four examinations , spiral ct scans covering the entire thorax were obtained with a somatom plus - s scanner ( siemens medical systems , erlangen , germany ) .
the parameters were a 3-mm collimation , 1-to-1 pitch , 1-sec scan time , 120 kvp , 165 ma , and a reconstruction interval of 2 mm .
for the measurement of cross - sectional area ( csa ) , we used the region - of - interest ( roi ) stat program , built into the ge 9800 quick scanner , by drawing a circle around selected bronchial lumen .
measurements were taken at four sites : both diaphragmatic bronchi and the lower portion of these bronchi that could be unambiguously identified at the same anatomic level under all experimental conditions ( fig .
the image data obtained from the somatom plus - s were transferred to an allegro medical imaging workstation ( isg technologies inc . ,
canada ) , and using a seeded volume of interest ( voi ) technique , csas were calculated ( 16 ) .
measurements were obtained at ten sites : the main , apical , diaphragmatic , and lower portion of diaphragmatic bronchi at both sides , the right diaphragmatic bronchus after the azygos , and the azygos bronchus ( fig .
when the operator set both upper and lower thresholds and defined a seed point in the selected bronchial lumen , an isodense area was automatically filled and the corresponding csa was calculated .
if a portion of the voi leaked into an area that we did not wish to include , the portion could be cut off by drawing a line at the point of leakage .
window width/ level were fixed at 1350 hu/ -450 hu , which were considered suitable for airway observation ( 10 - 11 ) .
we selected longitudinal airways , which were round . to compensate for errors due to slight differences in location at the selected bronchial site ,
csa was measured two to five times at each site , depending on the length and direction of each bronchus , and averaged . in four examinations in which spiral ct
was performed , 3-d reconstruction by means of the shaded surface - display technique was performed , using the data transferred to the allegro medical imaging workstation .
the volume data included from round tracheal lumen to the smallest bronchial lumen . using the seeded voi technique , as explained above , proximal airway volume ( pav )
the mean csa at each bronchial site represents the average of measured csas of nine examinations .
percentage changes occurring in mean csa , pav , and pap during the methacholine challenge and delayed state were represented as percentages of the control state , while those occurring during the ventolin challenge state were expressed as percentages of the delayed state , which provided a baseline value .
using wilcoxon 's signed rank test , changes in mean csas , suggesting airway reactivity , and in pav and pap after the administration of drugs , were statistically analyzed . by means of linear regression analysis , we correlated degree of airway activity , expressed as the percentage change induced by methacholine , with airway size , expressed as csa during the control state .
initially we performed a pilot study with a yorkshire pig model , but because thin - section ct showed that the bronchi of this animal were relatively thick - walled and small - calibered , and the response to airway stimulating agents was thus minimally visible , the model was rejected as unsuitable .
these animals have relatively thin - walled and large - calibered bronchi , and ct thus demonstrates airway reactivity ; they are , in addition , relatively easy to handle .
eight mongrel dogs weighting 10 - 12 kg underwent fasting for 6 hours prior to anesthesia , and atropine ( daehan atropine ; daehan pharmaceutical , seoul ) was injected intramuscularly .
the dogs were anesthetized by intramuscular injection of 0.2 ml / kg of a mixture of 1ml of 2% xylazine hydrochloride ( rompun ; bayer korea , seoul , korea ) and 1ml of 10% ketamine hydrochloride ( ketara ; yuhan yanghang , seoul , korea ) .
anesthesia was maintained by intravenous injection of 5 - 7 mg / kg of thiopental sodium ( pentothal ; choong wae pharmacy , seoul , korea ) . immediately after the induction of anesthesia
, intubation was performed using a 7.5-mm inside - diameter endotracheal tube and a bear-2 adult volume ventilator ( bear medical systems corporation , riverside , cal . )
provided ventilation at a rate of 20 breaths/ minute and a tidal volume of 300 ml . to minimize motion artifacts and achieve constant lung volumes during ct scanning ,
the animals were paralyzed by intravenous injection of 1 mg/ kg of succinylcholine chloride ( quelin ; abbott laboratories , chicago , ill . ) at functional residual capacity ( 11 ) .
two examinations were performed at an interval of two weeks . it has been reported that intravenous methacholine induces constriction only in the airways , whereas aerosolized methacholine causes constriction both in the airways and in the parenchyma ( 12 - 13 ) , and to induce bronchoconstriction , intravenous bolus injection of 0.1 mg / kg methacholine chloride ( provocholine ; roche , u.s.a . ) was therefore performed . to induce bronchodilation ,
2 ml of 1% salbutamol ( ventolin ; glaxowellcome ) was inhaled for 1 minute through a nebulizer connected to the ventilator . in our study , the procedure adopted by amirav et al .
( 15 ) was modified in such a way that initially , each dog underwent control thin - section ct , and after subsequent methacholine injection , the second series of ct scans was immediately obtained . after a 5-min delay ,
five minutes later , the final series of scans was obtained . in order to demonstrate the relationship between morphologic airway reactivity and physiologic parameters , peak airway pressure ( pap ) was measured through a pressure gauge connected to the ventilator immediately before ct scanning .
during the early period when spiral ct was not available , a ct / t 9800 quick scanner ( general electric medical system , milwaukee , wis . )
the number of slices was five to seven , each 3 cm below the tracheal carina and just above the diaphragm with an interval of 3-mm .
scanning parameters were 1.5-mm collimation , 1.5-mm interslice gap , 140 kvp , 170 ma and 1.5-sec scan time .
for the last four examinations , spiral ct scans covering the entire thorax were obtained with a somatom plus - s scanner ( siemens medical systems , erlangen , germany ) .
the parameters were a 3-mm collimation , 1-to-1 pitch , 1-sec scan time , 120 kvp , 165 ma , and a reconstruction interval of 2 mm .
for the measurement of cross - sectional area ( csa ) , we used the region - of - interest ( roi ) stat program , built into the ge 9800 quick scanner , by drawing a circle around selected bronchial lumen .
measurements were taken at four sites : both diaphragmatic bronchi and the lower portion of these bronchi that could be unambiguously identified at the same anatomic level under all experimental conditions ( fig .
the image data obtained from the somatom plus - s were transferred to an allegro medical imaging workstation ( isg technologies inc . ,
canada ) , and using a seeded volume of interest ( voi ) technique , csas were calculated ( 16 ) .
measurements were obtained at ten sites : the main , apical , diaphragmatic , and lower portion of diaphragmatic bronchi at both sides , the right diaphragmatic bronchus after the azygos , and the azygos bronchus ( fig .
when the operator set both upper and lower thresholds and defined a seed point in the selected bronchial lumen , an isodense area was automatically filled and the corresponding csa was calculated .
if a portion of the voi leaked into an area that we did not wish to include , the portion could be cut off by drawing a line at the point of leakage .
window width/ level were fixed at 1350 hu/ -450 hu , which were considered suitable for airway observation ( 10 - 11 ) .
we selected longitudinal airways , which were round . to compensate for errors due to slight differences in location at the selected bronchial site ,
csa was measured two to five times at each site , depending on the length and direction of each bronchus , and averaged .
in four examinations in which spiral ct was performed , 3-d reconstruction by means of the shaded surface - display technique was performed , using the data transferred to the allegro medical imaging workstation .
the volume data included from round tracheal lumen to the smallest bronchial lumen . using the seeded voi technique , as explained above , proximal airway volume ( pav )
the mean csa at each bronchial site represents the average of measured csas of nine examinations .
percentage changes occurring in mean csa , pav , and pap during the methacholine challenge and delayed state were represented as percentages of the control state , while those occurring during the ventolin challenge state were expressed as percentages of the delayed state , which provided a baseline value .
using wilcoxon 's signed rank test , changes in mean csas , suggesting airway reactivity , and in pav and pap after the administration of drugs , were statistically analyzed . by means of linear regression analysis , we correlated degree of airway activity , expressed as the percentage change induced by methacholine , with airway size , expressed as csa during the control state .
ct images revealed that the mean csas at multiple bronchial sites of the bronchi on changed after the injection of methacholine and the inhalation of ventolin ( table 1 , fig .
the mean csas of selected bronchi , as shown by ct , ranged from 9.6 to 101.8 ( median ; 32.9 ) mm . immediately after the injection of methacholine , mean csas of the bronchi at all sites significantly decreased ( change , 70.26.2% ( meanstandard deviation ) ; p = 0.002 ) . during the 5-min delayed state , csas were close , but still significantly different , to those observed during the control state ( change , 92.36.5% ; p = 0.009 ) .
five minutes after the inhalation of ventolin , mean csas significantly increased compared to those seen during the delayed state ( change , 113.111.4% ; p = 0.002 ) ( fig .
three - dimensional reconstruction images obtained in four dogs showed that the median value of pav decreased from 19270 mm during control state to 15801 mm immediately after the injection of methacholine ( change , 83.03.0% ) . during the 5-minute delay ,
the median value of pav increased to 18497 mm ( change , 92.46.5% ) , and five minutes after the inhalation of ventolin increased to 21084 mm ( change , 113.18.6% ) ( fig .
4 ) . during no sequence were the changes in pav statistically significant , however .
pap also changed after the injection of methacholine and the inhalation of ventolin ( table 2 , fig .
its level increased significantly from 15.41.0 cm h2o during the control state to 23.32.2 cm h2o immediately after the injection of methacholine ( change , 149.821.1% ; p = 0.003 ) . during the 5-minute delay ,
pap decreased to 16.31.1 cm h2o ( change , 105.95.2% ) , a significantly different level from that seen during the control state ( p = 0.03 ) .
five minutes after the inhalation of ventolin , pap rather increased to 16.61.5 cm h2o ( change , 100.74.4% ) , but this change was not statistically significant ( p = 0.9 ) .
although the absolute difference in csas induced by methacholine challenge was much greater in large airways than in small ones ( p = 0.0001 ) , the degree of airway reactivity , expressed as percentage change of in csas , did not correlate with airway size during the control state [ (regression coefficient ) = 0.01 ; p = 0.71 ] .
ct images revealed that the mean csas at multiple bronchial sites of the bronchi on changed after the injection of methacholine and the inhalation of ventolin ( table 1 , fig .
the mean csas of selected bronchi , as shown by ct , ranged from 9.6 to 101.8 ( median ; 32.9 ) mm . immediately after the injection of methacholine , mean csas of the bronchi at all sites significantly decreased ( change , 70.26.2% ( meanstandard deviation ) ; p = 0.002 ) . during the 5-min delayed state , csas were close , but still significantly different , to those observed during the control state ( change , 92.36.5% ; p = 0.009 ) .
five minutes after the inhalation of ventolin , mean csas significantly increased compared to those seen during the delayed state ( change , 113.111.4% ; p = 0.002 ) ( fig .
three - dimensional reconstruction images obtained in four dogs showed that the median value of pav decreased from 19270 mm during control state to 15801 mm immediately after the injection of methacholine ( change , 83.03.0% ) . during the 5-minute delay ,
the median value of pav increased to 18497 mm ( change , 92.46.5% ) , and five minutes after the inhalation of ventolin increased to 21084 mm ( change , 113.18.6% ) ( fig .
4 ) . during no sequence were the changes in pav statistically significant , however .
pap also changed after the injection of methacholine and the inhalation of ventolin ( table 2 , fig .
its level increased significantly from 15.41.0 cm h2o during the control state to 23.32.2 cm h2o immediately after the injection of methacholine ( change , 149.821.1% ; p = 0.003 ) . during the 5-minute delay ,
pap decreased to 16.31.1 cm h2o ( change , 105.95.2% ) , a significantly different level from that seen during the control state ( p = 0.03 ) .
five minutes after the inhalation of ventolin , pap rather increased to 16.61.5 cm h2o ( change , 100.74.4% ) , but this change was not statistically significant ( p = 0.9 ) .
although the absolute difference in csas induced by methacholine challenge was much greater in large airways than in small ones ( p = 0.0001 ) , the degree of airway reactivity , expressed as percentage change of in csas , did not correlate with airway size during the control state [ (regression coefficient ) = 0.01 ; p = 0.71 ] .
in this study , we were able to demonstrate by means of ct the extent to which bronchial reactivity was induced by both bronchoconstrictor and bronchodilator .
csa decreased significantly after the administration of methacholine , but a scan obtained five minutes later showed insufficient normalization .
methacholine induced a significant increase in pap , but again , the normalization observed after five minutes was insufficient .
the changes in pap induced by ventolin were not statistically significant , and we can speculate that after the five - minute delay , the methacholine effect had not completely disappeared .
methacholine has both a rapid onset ( < 2 mins ) and a relatively long duration of action ( at least 30 mins ) , and at the time of ventolin inhalation , both the bronchodilator effect of ventolin itself and a possible decrease in the effect of methacholine could have induced airway dilatation . after the inhalation of ventolin ,
pap did not significantly decrease despite the significant bronchodilation visible on ct , including that seen in a dog which showed an unexplainable increase in pap ( table 2 , examination 1 ) .
the effect of ventolin on the remaining dogs was that airway pressure either decreased or remained the same ( table 2 ) .
we believe that this discrepancy between the changes occurring in csa and in pap was due to the nonuniform deposition of aerosolized particles and discordance between morphology and physiology .
these particles , which are predominantly deposited in large airways , act only on those parts of the airway in which they are deposited ( 17 ) .
furthermore , since it is influenced much more severely by changes in small airways ( 18 ) , pap might not significantly decrease , even though sufficient bronchodilation of large airways was achieved by the amount of ventolin we used .
we believe , in addition , that the changes in airway areas revealed by ct , rather than changes in airway pressure , more sensitively reveal the bronchodilation induced by ventolin .
this study of the relationship between degree of airway reactivity and airway size was a trial for investigating local response at each bronchial level .
drazen and schneider ( 19 ) demonstrated a substantial decrease in the density of cholinergic receptors between trachea and lung parenchymal strips in the guinea pig .
nadel et al . ( 20 ) and shioya et al . ( 21 ) , on the other hand , observed that maximum constriction in airways , expressed as percentage change in diameter , occurred in smaller bronchi .
we observed that the percentage decrease in cross - sectional areas did not correlate with airway size , though absolute differences in area were larger in larger bronchi .
we speculate that the averaging of heterogeneous response in individual dogs led to a statistically insignificant result . in bronchi at the same level , individual dogs in our study demonstrated bronchoconstriction ranging widely from 40 to 80% of the control value .
herold et al . ( 11 ) believed that heterogeneity among dogs represented variation in individual responsiveness to bronchoconstrictor challenge .
physiologic studies to assess bronchial reactivity to stimuli provide information regarding overall response of the airway , but not on local response at each bronchial level , and morphologic as well as physiologic analysis may be valuable in assessing the degree of airway reactivity or the site of action of therapeutic agents . since the trial using tantalum bronchography in the study by nadel et al .
( 20 ) of the effect of vagal stimulation on airways , several reports have described the physiologic imaging of airways affected by bronchoconstrictor .
benson et al . ( 22 ) directly visualized airway response to vagal stimulation and inhaled histamine by using tantalum bronchography in their experiment involving dogs .
they were able to directly visualize changes in airway caliber , an approach which was more comprehensive than tests of forced expiration or measurements of total airway resistance .
in contrast to tantalum bronchography , which itself can provoke bronchoconstriction ( 11 ) , ct can demonstrate changes in the diameter of the central airways without additional stimulation of the bronchi , and human studies involving normal and asthmatic patients are thus possible safely .
high - resolution ct ( hrct ) can demonstrate bronchioles with a diameter of 2 mm or more , or with a wall thickness of more than 0.1 mm ( 23 ) .
the clinical application of hrct to airway diseases has been limited to static studies for the evaluation of fine structural abnormalities occurring in bronchiectasis , bronchiolitis , bronchial tumors , bronchial stenosis and congenital anomalies of the airways ( 24 ) .
the use of hrct as a tool with which to assess dynamic response of the lung to various stimuli has recently been introduced and actively investigated . using a dog model , brown et al .
( 10 ) and herold et al . ( 11 ) reported the hrct findings of the airways after the administration of bronchoconstrictor , demonstrating decreased csa of the bronchi after the inhalation of histamine .
bronchoconstriction has been induced by the inhalation of methacholine or histamine , and in normal subjects , it has been suggested that the predominant site for bronchoconstriction by methacholine is the large airways , while histamine constricts the small airways around the alveolar ducts and alveoli in normal subjects via its direct action ( 4 ) .
we used methacholine in this study because of its preferential constricting effect on the large airways , visible with thin - section ct .
although it has been suggested that the respiratory tract is most reactive to nebulized methacholine ( 25 ) , we administered the drug intravenously because the sites and rates of deposition of aerosolized particles are determined by the particle size of aerosols , and the frequency and tidal volume of respiration . due to its commercial availability
, we used ventolin ( salbutamol ) as ( 2-agonist acting on both the central and peripheral airways in subjects with asthma ( 6 ) , and most commonly inhaled .
volumetric acquisition by means of spiral ct and 3-d volume rendering of the proximal airway , performed in four dogs in our study , showed results similar to those obtained by axial ct imaging .
because it reflects not only transverse but also longitudinal changes , the measurement of airway volume using volumetric ct data might be more accurate in the evaluation of airways than measurement involving axial images . according to a phantom study performed to determine the accuracy of the method by which the length , diameter , and branch angles of pulmonary trees are measured using ct data
, thin - section ct and volumetric spiral ct with 3-d reconstruction can clearly demonstrate airway reactivity to bronchostimulator . in our limited study
, the degree of such reactivity revealed by thin - section ct did not correlate with airway size .
ct scanning can therefore be used in the evaluation of pathophysiologic response of the airways .
 objectiveto determine the extent to which thin - section and volumetric three - dimensional ct can depict airway reactivity to bronchostimulator , and to assess the effect of different airway sizes on the degree of reactivity.materials and methodsin eight dogs , thin - section ct scans were obtained before and after the administration of methacholine and ventolin . 
 cross - sectional areas of bronchi at multiple levels , as shown by axial ct , proximal airway volume as revealed by three - dimensional imaging , and peak airway pressure were measured . 
 the significance of airway change induced by methacholine and ventolin , expressed by percentage changes in cross - sectional area , proximal airway volume , and peak airway pressure was statistically evaluated , as was correlation between the degree of airway reactivity and the area of airways.resultscross-sectional areas of the bronchi decreased significantly after the administration of methacholine , and scans obtained after a delay of 5 minutes showed that normalization was insufficient . 
 ventolin induced a significant increase in cross - sectional areas and an increase in proximal airway volume , while the effect of methacholine on the latter was the opposite . 
 peak airway pressure increased after the administration of methacholine , and after a 5-minute delay its level was near that of the control state . 
 ventolin , however , induced no significant decrease . 
 the degree of airway reactivity did not correlate with airway size.conclusionthin-section and volumetric spiral ct with three - dimensional reconstruction can demonstrate airway reactivity to bronchostimulator . 
 the degree of reactivity did not correlate with airway size . 
MATERIALS AND METHODS
Selection of Animal
Animal Preparation
Study Design
CT Examination
Measurement of Cross-Sectional Area
Volumetry
Data Analysis
RESULTS
Changes in Mean CSAs and PAV after the Administration of Drugs
Correlation between Degree of Airway Reactivity and Airway Size
DISCUSSION