smoke-exposed animals exhibited evidence of hyperresponsiveness. In this ... animals to cigarette smoke is associated with the development of bronchial.
Lung (1993) 171:95-107
Airway Hyperresponsiveness in Cigarette Smoke-Exposed Rats L. J. Xu, I R. J. Dandurand, 2 M. Lei, ~ and D. H. Eidelman 3 IMeakins-Christie Laboratories, McGill University 3626 St. Urbain Street, McGill University Clinic, Royal Victoria Hospital, 2Montreal Chest Hospital Centre, 3Montreal General Hospital, McGill University, Montreal, Quebec, H2X 2P2 Canada
Abstract. To investigate the possibility that altered airway-parenchymal interaction may account for bronchial hyperresponsiveness induced by cigarette smoke exposure, we tested the effect of administration of cigarette smoke (SM), elastase (EL), and both SM and EL on airway responsiveness in 41 Long-Evans male rats. Twelve were exposed to 30 puffs of SM for 15 weeks; 8 received a single intratracheal injection of EL (250 IU/kg); 9 received both EL and SM exposure (SE); 12 control rats were exposed to room air (CO). After 15 weeks, animals were anesthetized and mechanically ventilated (Vt = 2.5 ml, f = 80/rain). Methacholine (MCh) dose-response curves (DRCs) were constructed by calculating pulmonary resistance (RL) after ultrasonic nebulization of saline followed by doubling concentrations of MCh (0.0625-256 mg/ml). Exposure to cigarette smoking, with or without elastase, led to a significant reduction in body weight and increased total lung capacity (TLC) compared to exposure to CO. However, there was no significant change in static compliance in the experimental groups, despite increased lung volume. The concentration resulting in a doubling of RL (EC200RL) was significantly lower in rats treated with SM (n = 7) than CO (n = 8) (3.3 vs. 56.1 mg/ml, geometric mean, p < 0.01). The concentration at which a maximal RL was achieved was lower in SM than CO, EL, and SE (p < 0.05). To assess the possible influence of airway-parenchymal interaction on responsiveness, we measured RI~ both at functional residual capacity (FRC) and at a volume above FRC equivalent to 1 tidal volume. RL changed similarily in all groups. Despite similar effects on mechanics of both cigarette smoke exposure and elastase administration, only cigarette smoke-exposed animals exhibited evidence of hyperresponsiveness. In this model cigarette smoke-induced hyperresponsiveness is unrelated to changes in either lung elasticity or airway-parenchymal interaction.
Offprint requests to: D. H. Eidelman
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L.J. Xu et al. Key words: Hyperresponsiveness--Cigarette smoke--Elastase--Interdependence.
Introduction Several investigators have observed that relatively short exposures of small animals to cigarette smoke is associated with the development of bronchial hyperresponsiveness [7, 9, 14, 15], although the mechanism of this is unknown [15]. In humans, chronic cigarette exposure is associated with disruption of parenchymal attachments, even before the development of overt emphysema [ 17]. Bellofiore and co-workers [ 1] have reported that disruption of parenchymal attachments by elastase administration to rats leads to mechanical uncoupling between the airways and the parenchyma and the development of bronchial hyperresponsiveness [1], consistent with the notion that responsiveness is in part determined by mechanical factors. We wished to investigate the possibility that cigarette-induced hyperresponsiveness in an animal model might relate to parenchymal damage, particularly at the level of the parenchymal attachments to airways, and consequent changes in airway-parenchymal interaction. Furthermore, we wondered if the combination of cigarette smoke exposure and low-dose elastase administration, which has been described to result in emphysema in the rat [11], might interact to result in a greater degree of responsiveness than that resulting from cigarette exposure alone. To investigate these questions we exposed Long-Evans rats to cigarette smoke, elastase, and a combination of cigarette smoke and elastase and evaluated bronchial responsiveness using methacholine (MCh) dose-response curves. To assess specifically the possible role of airway-parenchymal interaction on responsiveness, we also investigated the effect of increasing FRC on pulmonary resistance during MCh exposure [1]. Under normal circumstances, small changes in FRC may strongly influence measurements of pulmonary resistance [1, 5]. We reasoned that if airway-parenchymal coupling was altered by cigarette smoke or elastase, this would be reflected in abolition of the effect of lung volume change on pulmonary resistance. Our results confirm that cigarette smoke exposure in the rat is associated with hyperresponsiveness, but do not support the hypothesis that this is related in any way to alterations in pulmonary mechanics.
Materials and Methods Animals
Forty-one male Long-Evansrats weighingapproximately250 g were obtainedfrom Charles River (Montreal, QC). Four groups of animals, 8-12 animals per group, were studied concurrently. Control (CO) rats (n - 12) were exposedto shamsmoking30 rain/day, 5 clays/week,for 15 weeks.
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Rats exposed to cigarette smoke (SM, n = 12) and those exposed to both cigarette smoke and elastase (SE, n = 9) were exposed to the smoke of 3 cigarettes per day, 5 days per week, for 15 weeks. Both EL rats (n = 8) and SE rats received 0.5 ml of 0.15 N saline containing purified porcine pancreatic elastase (250 IU/kg; Sigma [St. Louis, MO]) by intratracheal instillation. EL rats then underwent sham smoking for 15 weeks.
Cigarette Smoke Administration Cigarette smoke was administered using a modification of the method employed by Kimmel and colleagues [11]. A peristaltic pump was used to generate puffs of mainstream cigarette smoke or air, which was delivered to an inhalation chamber. Puffs were of approximately 20 sec duration. Four rats at a time were exposed to cigarette smoke or air. Each rat was placed in a cylindrical tube designed to keep the rat's shout in proximity to the source of cigarette smoke. Rats were exposed in a single session, 5 days/week, to the smoke of 3 cigarettes/day. Each cigarette took 15 min to burn, with a 10 min break between cigarettes. Cigarettes were obtained from a commercial supplier (Export A brand, Imperial Tobacco [Montreal, Canada]).
Lung Mechanics Lung mechanics were measured at end of the 15 weeks as follows: animals were anesthetized wth urethane (50%, 1 mg/kg, intraperitoneally [i.p.]), and intubated with an 11 cm polythene tracheal cannula (PE 240) through a tracheostomy. Esophageal pressure (Pes) was measured using a salinefilled polyethylene catheter (PE 90, 30 cm long) placed in the lower esophagus in such a way as to maximize cardiac oscillations and connected to 1 port of a differential pressure transducer (Sanborn 367B, Hewlett-Packard, [Waltham, MA]). The other port of the pressure transducer was connected via a side arm to the tracheal catheter. Transpulmonary pressure (P0 was obtained as the output of the differential pressure transducer. All physiological measurements were carried out in a pressure plethysmograph. Volume was measured with a pressure transducer (Validyne MP-45 -+ 100 cmH20) calibrated for volume change. The plethysmograph consisted of 2 cylindrical Plexiglas chambers (3 1 in total volume) interconnected by a narrow Plexiglas tube (3 cm ID, 3.5 cm long). One chamber was occupied by the animal while the other was filled with copper mesh to reduce adiabatic effects and temperature-related pressure changes. Airflow ('q) was obtained by differentiation of volume. Baseline RL and.EL were determined as follows. The V L signal was numerically differentiated to calculate V. V D V, and PL signals were fitted to the following equation using multiple linear regression: PL = ELVL + R L ~" + K
(1)
where EL is pulmonary elastance, R L is pulmonary resistance, and K represents the end-expiratory pressure as welt as any error in fitting the equation to the data. The signals were conditioned using antialiasing filters and were sampled at 200 Hz using an A/D board (DT-2801-A, Data Translation [Marlboro, MA]) installed in a microcomputer (Compaq Deskpro 286 [Compaq Co., Houston, TX]). Functional residual capacity (FRC) was determined by the method of Dubois and co-workers [6] by occluding the airway opening at end-expiration and determining the relationship between changes in V L and Pes during consecutive occluded respiratory efforts. The average of 3 determinations was obtained in each rat. Total lung capacity (TLC) was taken as the lung volume coresponding to a PL of 30 cmH20. Static deflation pressure-volume curves were obtained following 3 inflations to TLC to standardize volume history and suppress spontaneous respirations. A further inflation to TLC was held for 2-3 sec and deflation was allowed to occur passively over 3-5 sec, during which serial
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interruptions of expiratory flow were made at 1 mt decrements. This maneuver was performed 3 times while PL and V L were sampled continuously. The equation V=A-
Be KP
(2)
was fitted to the data from FRC to TLC using the iterative least-squares method [3]. In Equation 2, V represents volume, P transpulmonary pressure, and A, B and K are constants. K was used as an index of curvature of the fitted curve [4]. Fitted curves were used to calculated compliance (Cst) between FRC and FRC plus 0.5 ml, and specific compliance (Csp) as Cst divided by FRC.
Methacholine Challenge Baseline R L, FRC, and pressure-volume curves were measured while the rats were spontaneously breathing. The rats were then paralyzed by injection of pancuronium bromide (1 mg/kg, i.p.) and mechanically ventilated (Vt = 2.5 ml, f = 80/min). Aerosols were delivered by an ultrasonic nebulizer (Ultra-Neb 100 [DeVilbiss, Somerset, PA]) into the intake port of the ventilator. The nebulizer produces particles with a mean aerodynamic diameter of 4.5-6.0/z. An airflow of 11 ml/sec was used with a nebulizer output of 0.18 ml/min. Aerosols were delivered for 30 sec, starting with saline and continuing with progressively doubling concentrations of MCh ranging from 0.0625 to 256 mg/ml. R L and Ec were determined twice after saline and each concentration of MCh both at FRC and at a V c corresponding to PL 2 cmH20 above FRC.
Volume Maneuver V L was increased by applying continuous suction to a port of the plethysmograph. The level of suction was adjusted by a valve in parallel with the wall suction through which the rate of room air entrainment could be controlled. A continuous tracing of PL was displayed on the microcomputer screen while the valve was closed sufficiently to raise Pc by 2 cmH20. This level of suction was applied for 30 sec, the suction line to the plethysmograph was clamped, and a second determination of RL and Ec was obtained. The time interval between sampling signals at the 2 different lung volumes was approximately 60 sec. The plethysmograph was vented back to atmospheric pressure. The next aerosol was administered and the cycle repeated for each concentration of MCh.
Responsiveness Indices Responsiveness was expressed as the MCh concentration required to increase R L to 200% of the value measured after saline aerosolization (EC200Rc), calculated by linear interpolation between bracketing concentrations of MCh. E C m a x w a s the concentration resulting in a maximal RL.
Morphometric Studies Immediately after the MCh concentration-response curve data were collected, the animals were exsanguinated and the lungs were removed, distended, and perfused intrabronchially with 10% neutral buffered formalin at a constant pressure of 30 cmH20 for 48 hr. A midsagittal section cut from each right lung was processed for histologic studies. The lung tissue was embedded in paraffin, cut into 5 ~m thick sections, and stained with hematoxytin-phloxine-safran (HPS). The right lung was used for all morphometric studies.
Airways The airways were assessed for pathologic change using a semiquantitive scoring system adapted from Renzi and co-workers [16]. The extent of airway edema, epithelial detachment, and cellular
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infiltration was scored as follows. First, each individual airway was evaluated and the presence or absence of the pathologic change recorded. A score of 0 was given if none of the airways had the change looked for, 1 + if >0 but -< 25%, 2 + if >25% but -< 50%, 3 + if >50% but -< 75%, and 4 + if more than 75% had the change sought.
Mean Linear Intercept Mean linear intercept (Lm) was measured in 20 randomly selected fields from each lung using a calibrated eyepiece according to the method of Thurlbeck [19]. No correction for shrinkage was made.
Attachments Attachments to membranous intraparenchymal airways were counted and evaluated for breaks using the method of Saetta [17]. Results are expressed as attachments/mm of airway wall (ATT) and number of abnormal alveolar attachments (AA) as a percentage of total attachments per airway. Airway size was measured by projecting the image of airways onto a computer-controlled digitizing board (Jandel Scientific [Corte-Madera, CA]) using a microscope drawing tube attachment. As described by Saetta [17], we measured the external perimeter of the airways to permit calculation of numbers of airways/mm. Only airways with ratios of long to short axis less than 2 : 1 were evaluated.
Data Analysis Group results are expressed as mean _+ SD. Differences between group means were tested for significance using 1 way analysis of variance (ANOVA). Post hoc analysis of differences was done using the Newman-Keuls test. A significant difference was said to be present when p < than 0.05. All statistical analyses were made using the NCSS statistical package (NCSS [Kaysville, UT]). A plateau in the concentration-response curve is defined as no increase in R L for at least 3 consecutive concentrations.
Results
Body Weight and Markers of Smoke Exposure B o d y w e i g h t at t h e s t a r t o f t h e e x p e r i m e n t d i d n o t d i f f e r b e t w e e n g r o u p s ( T a b l e 1). A t t h e e n d o f 15 w e e k s , t h e m e a n b o d y w e i g h t s o f t h e C O a n d E L - t r e a t e d g r o u p s w e r e s i g n i f i c a n t l y h i g h e r t h a n t h o s e o f g r o u p s S M a n d S E ( T a b l e 1). Blood levels of percentage total hemoglobin (COHb) averaged over the t o t a l e x p o s u r e p e r i o d w e r e s i g n i f i c a n t l y i n c r e a s e d in S M (13.45 -+ 3.67, n = 4) a n d S E (8.70 -+ 3.09, n = 4) c o m p a r e d to C O (3.74 -+ 0.77, n = 5) a n d E L t r e a t e d a n i m a l s (2.83 -+ 1.81, n = 3); p < 0.001.
Lung Volume T h e r e s u l t s o f p u l m o n a r y f u n c t i o n t e s t s f o r all g r o u p s a r e s h o w n in T a b l e 1. T L C w a s s i m i l a r in all g r o u p s . H o w e v e r , a f t e r w e n o r m a l i z e d b y B W , t h e T L C
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Table 1. Body weight, static lung volumes, and pulmonary mechanics
BW (g) baseline BW (g) 15 weeks FRC (ml) FRC/BW (ml/kg) TLC (ml) TLC/BW (ml/kg) Cst (ml/cmH20 I) Csp (cmH20l) K (cmH20 l) *
CO (n = 12)
SM (n = 12)
EL (n = 8)
SE (n = 7)
293.3 (34.84)
301.0 (18.44)
293.8 (41.52)
265.1 (20.20)
557.0 (68.82)
461.5 (50.15)+*
568.7 (50.51)
433.2 (63.67)+*
6.14 (1.88) 10.96 (3.30)
7.32 (2.87) 15.88 (6.55)
7.51 (2.30) 13.10 (4.54)
6.83 (1.94) 15.46 (3.89)
19.51 (2.58) 35.02 (5.53)
20.16 (3.38) 43.73 (8.48)*
22.85 (2.86) 39.50 (3.03)
21.03 (1.79) 48.38 (6.78)+ *
2.40 (0.90)
2.00 (0.82)
2.57 (0.96)
2.59 (1.07)
0.46 (0.37)
0.32 (0.18)
0.34 (0.13)
0.43 (0.25)
0.18 (0.06)
0.15 (0.06)
0.16 (0.06)
0.18 (0.07)
Values are mean -+ SD. p < 0.001 when compared to CO; + p < 0.001 when compared to EL.
was significantly greater in SM and S E c o m p a r e d to C O - t r e a t e d g r o u p s (Table I, p < 0.01). T h e T L C o f S E was also significantly greater than E L - t r e a t e d g r o u p s (Table 1, p < 0.01). T h e r e was a t e n d e n c y for the F R C o f all 3 experimental g r o u p s to be i n c r e a s e d c o m p a r e d to the CO group, a l t h o u g h this did not r e a c h statistical significance after we normalized data for b o d y weight (Table 1).
Lung Recoil Static deflation p r e s s u r e - v o l u m e c u r v e s (PV) for all the groups are s h o w n in Fig. 1. T h e P V c u r v e s in SM, SE, and E L - t r e a t e d g r o u p s w e r e shifted to higher v o l u m e s c o m p a r e d to controls, especially after we n o r m a l i z e d data for b o d y weight. This primarily reflected the c h a n g e s in b o d y weight. T h e indices o f P V c u r v e shape, Cst, C s p and K, w e r e similar in the 4 g r o u p s (Table 1).
Airway Responsiveness Fig. 2 s h o w s the m e a n c o n c e n t r a t i o n - r e s p o n s e c u r v e (CRC) to M C h p e r f o r m e d in the 4 g r o u p s at 2 lung v o l u m e s . Rc did not r e a c h a plateau in the CO, b u t p l a t e a u x in Rc w e r e seen in SM (6/7), E L (4/8), and S E - t r e a t e d g r o u p s (2/8) b e t w e e n 16 and 256 mg/ml. T h e c o n c e n t r a t i o n - r e s p o n s e c u r v e s o f SM and SEtreated animals t e n d e d to be shifted leftward and to exhibit i n c r e a s e d m a x i m a l r e s p o n s i v e n e s s . ECz00R L was significantly l o w e r in SM (n = 7) and S E (n =
Airway Hyperresponsiveness in Smoke-Exposed Rats
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7) than CO groups (n = 8) (Fig. 3, 3.3 mg/ml vs. 56.1 mg/ml, geometric mean, p < 0.01). ECma x w a s significantly lower in SM than CO, EL, and SE-treated groups (43.5 mg/ml vs. 91.2 mg/ml, 117.2 mg/ml, and 128.0 mg/ml, respective geometric means, p < 0.005). Maximal R E was greater in SE (1.14 +- 0.12 cmH20/1/sec) and SM (1.09 -+ 0.38 cmH20/1/sec) than CO (0.88 -+ 0.15 c m H 2 0 / 1/sec), and EL-treated groups (0.98 -+ 0.29 cmH20/1/sec), (p < 0.05). In all groups the maximal value of R L was significantly reduced by increasing lung volume. The effect of increasing lung volume on R L was similar in all groups (Fig. 2). We specifically found no significant difference among groups in the decrease in R L at 256 mg/ml of MCh increasing PL by 2 cmH20 (CO, 0.173 _+ 0.203 cmH20; SM, 0.237 _+ 0.219 cmH20; EL, 0.224 -+ 0.101 cmH20; SE, 0.271 + 0.154 cmH20). Morphometry
Although there was a tendency for the L m to be increased in all of the treatment groups compared to CO, this did not reach statistical significance (Table 2). After we normalized data by length of basement membrane, the number of attachments was similar in all animals. Although some breaks in airway attachments were found in all animals, the percentage of broken attachments to total attachments was not different between groups (Table 2).
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L . J . Xu et al. 1.2 co (n=a)
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With regard to airway pathologic change, inspection of the slides revealed little evidence of active inflammation or damage. This is reflected in the airway score results (Table 3). Neither low-dose elastase exposure nor cigarette exposure had any effect on cellular infiltration, edema, or epithelial disruption of the airways. Discussion
The results of this study demonstrate that short-term exposure of rats to cigarette smoke, although insufficient of itself to induce pulmonary emphysema, leads to increased bronchial responsiveness to methacholine. We found no evidence of hyperresponsiveness to methacholine among rats treated with lowdose elastase alone and no evidence that elastase administration added to the hyperresponsiveness induced by smoking. Furthermore, we found no evidence
Airway Hyperresponsiveness in Smoke-Exposed Rats
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Fig. 3. Concentration of MCh required to increase pulmonary resistance (RE) to 200% of baseline value (EC~00Rk) in CO (open circles), SM (closed circles), EL (open triangles), and SE-treated groups (closed triangles). EC200RL was significantly lower in SM compared to CO-treated groups (p < 0.01).
Table 2. Morphometry
Lm (/zm) Attachments/ram Abnormal attachments (%)
CO (n = 12)
SM (n = 12)
EL (n = 8)
SE (n = 7)
88.8 (12.05) 14.77 (2.58) 12.49 (3.88)
90.80 (17.11) 16.28 (2.45) 12.22 (5.78)
96.84 (9.70) 15.56 (2.39) 13.36 (3.52)
102.70 (18.78) 16.28 (2.77) 14.12 (3.79)
Values are mean -+ SD. SM, EL, and SE had higher Lm than CO. The number of attachments/ mm are similar in 4 groups. There is no significant difference in percentage of abnormal attachments in 4 groups.
to implicate changes in airway-parenchymal interdependence as the mechanism of this hyperresponsiveness. The design of the study was similar to that previously described by Kimmel and colleagues [11], who used a combination of cigarette smoke exposure and low-dose elastase as a means of inducing emphysema. Although cigarette smoke exposure induced changes in body weight and carboxyhemoglobin levels as expected, we did not find evidence of emphysema morphologically despite levels of exposure to both eIastase and cigarette smoke similar to those employed by Kimmel et al. [11]. The differences between our results and theirs could be related to differences in smoking apparatus and cigarette type.
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L. J. Xu et al.
Table 3. Airway pathologic findings
Cellular infiltration Edema Epithelial disruption
CO (n = 8)
SM (n = 8)
EL (n = 7)
SE (n = 9)
1.63 (0.74)
1.50 (0.53)
1.29 (0.49)
1.33 (0.50)
2.88 (0.83) 1.50 (1.07)
1.00 (1.20) 1.62 (0.92)
2.29 (1.25) 1.29 (0.76)
1.67 (1.22) 1.00 (0.71)
Values are mean -+ SD. No significant differences were found between groups for cellular infiltration, edema, and epithelial disruption.
We found evidence of increases in TLC and FRC among both cigarette smoke-exposed and elastase-exposed groups of rats compared to control which reached significance when volumes were normalized for body weight (Table 1). Nevertheless, we found no significant changes in elastic recoil pressure or shape of the pressure-volume curve following exposure to elastase, cigarette smoke, or both (Fig. 1). These physiological findings are consistent with the morphologic observations. We did not see any definite evidence of emphysema in these rats and there were no significant differences in Lm among groups. However, there was a tendency for Lm to be increased in the cigarette smoke exposed groups, particularly in the group exposed to both cigarette smoke and elastase. In view of the previous report by Kimmel and colleagues [11], it is possible that a more prolonged or higher dose exposure to cigarette smoke coupled with elastase administration would have resulted in a significant increase in Lm. Despite the absence of parenchymal destruction, we did observe hyperresponsiveness to methacholine in cigarette smoke-exposed animals in which the dose-response curves were shifted leftward, and maximal responsiveness was increased (Fig. 2). This was reflected in the EC200RL, in the maximal resistance achieved during the study, as well as in the concentration of methacholine at which a maximal response occurred (Figs. 2, 3). In contrast, we did not observe any significant effect of low-dose elastase on responsiveness compared to controls (Fig. 2). Another prominent feature of the concentration-response curves was the detection of plateaux in RL in the concentration-response curves especially in group SM. Although it is possible that these plateaux may represent a cigarette smoke-induced change in factors limiting bronchoconstriction, the most likely explanation is that the plateaux were detected because the leftward shift of the concentration-response curves made it possible to reach maximal responses within the concentration range used. Drug solubility and systemic side effects are likely to have prevented us from using sufficiently high agonist concentrations to detect plateaus in group CO. In that the absence of a plateau is a
Airway Hyperresponsivenessin Smoke-ExposedRats
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hallmark of asthma [12], our finding of induced plateaux suggests that cigarette smoke-induced hyperresponsiveness differs in mechanism from that seen in asthmatic subjects. We originally hypothesized that hyperresponsiveness in this model resulted from an alteration in airway-parenchymal interaction leading to a reduction in mechanical load on airway smooth muscle [1, 12]. It has been argued that the lung parenchyma acts as a mechanical load against which airway smooth muscle shortening occurs. The forces of interdependence between the airway and parenchyma are such that the parenchyma may exert an outward force tending to keep the airways open. When airway smooth muscle shortens in response to a stimulus, such as methacholine administration, it must overcome the stress placed on the airway wall by the parenchyma. A number of authors have produced evidence that this mechanism may be an important determinant of responsiveness. Ding and colleagues have shown evidence that relatively small changes in FRC in normal humans can cause a large change in the dose-response curve [5]. Likewise, in cats Sly and colleagues have found evidence that lung volume can influence responsiveness [18]. Bellofiore and co-workers [1] administered relatively high doses of elastase to rats and demonstrated increased bronchial responsiveness to methacholine associated with evidence of experimental emphysema and destruction of parenchymal attachments to bronchioles. To investigate airway-parenchymal interaction, they measured pulmonary resistance at FRC and at a lung volume corresponding to approximately 1 tidal volume above FRC. Increasing FRC in control animals led to a fall in pulmonary resistance, which was abolished in the elastase-treated group. These findings were interpreted as evidence that the airways and parenchyma had become mechanically uncoupled. Our findings in cigarette smoke-induced hyperresponsiveness contrast with those of Bellofiore and co-workers [1]. We did not find any evidence that changing the resting lung volume significantly influenced dose-response curves in animals in whom cigarette smoke exposure led to hyperresponsiveness. Further, as noted above, we observed plateaux in RL following cigarette smoke exposure that were absent in the controls. This is opposite to what would have been expected if altered airway-parenchymal interaction accounted for the changes in the dose-response curve. The findings of hyperresponsiveness following cigarette smoke exposure in this study are similar to those reported in the guinea pig [7, 15]. It has been argued that in the guinea pig hyperresponsiveness may be a result of increased airway permeability [2, 7, 9]. We found no microscopic evidence of pathologic changes in the airways, but we did not measure airway permeability. In this regard, Nishikawa and colleagues, in a recent report of a dose-dependent effect of acute cigarette smoke exposure on bronchial responsiveness in the guinea pig, found no evidence of altered tracheal permeability or of neutrophil influx [15]. Likewise, although it has been suggested that cigarette smoke-induced bronchial hyperresponsiveness in humans may relate to changes in epithelial permeability [2, 9], Kennedy and co-workers [10] found no relationship between pulmonary epithelial permeability and baseline responsiveness in smokers. A1-
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though our data do not support a role for inflammatory processes in the generation of hyperresponsiveness in this model, inflammation that occurred early in the course of cigarette exposure, such as that described by Hulbert and colleagues [7] in the guinea pig [8], may be an important determinant of responsiveness. If neither airway inflammation nor airway-parenchymal interaction explains the phenomenon of cigarette smoke-induced bronchial hyperresponsiveness, it is necessary to consider other possible mechanisms. An alteration in baseline airway resistance could affect bronchial responsiveness. However, we found no differences in baseline resistance between groups. Another possibility relates to the potential role of cholinergic mechanisms, as suggested by the observation that increases in pulmonary resistance induced by cigarette smoke may be vagally mediated [13, I4]. It is possible that the cholinergic reflex pathway, or other neural pathways, are altered by cigarette exposure even in the absence of significant airways inflammation. In summary, we found that brief exposure of rats to mainstream cigarette smoke resulted in significant changes in the dose-response curve to methacholine, indicating hyperresponsiveness. However, these changes were not augmented by administration of low-dose elastase. There was no evidence that this form of hyperresponsiveness was related to changes in elastic recoil pressure, to airway-parenchymal interdependence, or to airway inflammation.
Acknowledgments. The authors wish to thank Ms. Barbara Kidd for her assistance in the preparation of the manuscript. We also thank suggestions. This work was supported by Research Fund. R. J. Dandurand D. H. Eidelman is an investigator
Dr. J. Martin for reviewing the manuscript and for his helpful the Quebec Thoracic Society and the J. T. Costelto Memorial was a recipient of a Canadian Lung Association Fellowship. of the Respiratory Health Network of Centres of Excellence.
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Accepted for publication: 14 September 1992
