Differences between inhaled and intravenous bronchial challenge to detect O3-induced hyperresponsiveness
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《应用生理学杂志》
Departamento de Investigación en Asma, Instituto Nacional de Enfermedades Respiratorias, and Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, CP 14080, México DF, México
ABSTRACT
Ozone (O3)-induced airway hyperresponsiveness in laboratory animals is usually demonstrated through dose-response curves with inhaled or intravenous bronchoconstrictor agonists. However, comparability of these two routes has not been well documented. Thus guinea pig airway responsiveness to ACh and histamine was evaluated 16-18 h after O3 (3 parts/million, 1 h) or air exposure by two plethysmographic methods (spontaneously breathing and mechanically ventilated) and by two administration routes (inhalatory or intravenous). We found that O3 caused airway hyperresponsiveness to intravenous, but not to inhaled, agonists, independent of the plethysmographic method used. Suitability of the inhalatory route to detect airway hyperresponsiveness was corroborated with inhaled ACh after an antigen challenge or extending O3 exposure to 3 h. Acetylcholinesterase activity was not modified after O3 exposure in lung homogenates and blood samples. Thus inhaled agonists were less effective to reveal the airway hyperresponsiveness after an acute O3 exposure than intravenous ones, at least for the 1-h exposure to 3 parts/million, and this difference seems not to be related to an O3-induced inhibition of the acetylcholinesterase activity.
keywords:ozone; airway hyperresponsiveness; dose-response curve; guinea pig
INTRODUCTION
EXPOSURE TO OZONE (O3) is a widely used method to induce airway hyperresponsiveness in different species, including guinea pigs and humans (3, 15, 20). The increased responsiveness is usually demonstrated through pharmacological methods, i.e., the elaboration of dose-response curves with bronchoconstrictor agents. In laboratory animals, the most common ways to deliver these agents to the airways are the intravenous (22, 23) and the inhalatory routes (14, 17). Although extensively used to demonstrate this phenomenon, differences between inhaled and intravenously administered agents have been very scantily investigated (18). This is an important issue because conclusions derived from experiments using one route might not be comparable with those using the other one, unless equivalence of both routes was assessed. In this sense, Corddry et al. (8) have demonstrated in dogs that whole lung resistance (RL) and peripheral airway resistance (Raw) measurements detected the same pattern of responsiveness to intravenous histamine (His), whereas they yielded different results with nebulized His. In the present work, we found that, at a certain O3 exposure magnitude, inhaled agonists did not detect airway hyperresponsiveness as the intravenous ones did, and some possible mechanisms for this difference are discussed.
MATERIALS AND METHODS
Animals and experimental design. Male Hartley guinea pigs (450-500 g body wt), bred in conventional conditions (filtered conditioned air, 21 ± 1°C, 50-70% humidity, sterilized bed) and fed with Purina pellets supplemented with disinfected fresh alfalfa and sterilized water, were used. Airway hyperresponsiveness to ACh and His was assessed by constructing dose-response curves. Two methods were used to measure the responses: plethysmography in spontaneously breathing (SB) animals and plethysmography in mechanically ventilated (MV) animals. In either method, bronchial challenges were accomplished by delivering the drugs by nebulized (NEB) or intravenous (IV) route. Therefore, four main groups of animals were integrated: SB/NEB challenge, SB/IV challenge, MV/NEB challenge, and MV/IV challenge.
With regard to the SB/NEB group, the noninvasive nature of the technique allowed us to perform two doses-response curves in the same guinea pigs. In these animals, the first curve was done ~24 h before the O3 or air (control group) exposure, and the second was done 16-18 h after such exposure. In the remaining groups, only one dose-response curve after air or O3 exposure was performed in each animal. In addition, a group of sensitized guinea pigs was submitted to a dose-response curve to inhaled ACh 24 h before and 3 h after an antigenic challenge. Finally, separate groups of guinea pigs with or without O3 exposure were used to evaluate the acetylcholinesterase (AChE) activity in lung homogenates and blood.
Plethysmography in spontaneously breathing animals. Each guinea pig was introduced into a plethysmographic chamber designed for unrestrained, freely moving animals (Buxco Electronics, Troy, NY). This chamber is supplied with a constant air flow (10 ml/s) that does not alter the respiratory signals. This methodology has been comprehensively described elsewhere (5, 6, 12). Briefly, the pressure inside the chamber was measured by a differential pressure transducer (SCXL004DN, Sen Sym, Milpitas, CA) connected to a preamplifier and continuously monitored through software (Buxco Biosystem XA, version 1.1). Changes in box pressure represent the difference between the thoracic expansion or contraction and the tidal volume (air removed from or added to the chamber during inspiration or expiration). The box pressure is differentiated to give a pseudoflow signal, which is then analyzed by the software to give an index named "enhanced pause" (Penh). The Penh value is obtained for each respiration by the following formula
where TE is expiratory time, RT is relaxation time, PEpeak is peak expiratory pressure, and PIpeak is peak inspiratory pressure. Penh values used in our study were obtained from averaging logged values every 15 s. The software was adjusted to only include breaths with a tidal value of 1 ml, with a minimal inspiratory time of 0.15 s, a maximal inspiratory time of 3 s, and a maximal difference between inspiratory and expiratory volumes of 10%. A recent study in mice (12) demonstrated that Penh had a close, positive correlation with the measurement of total RL, which in turn can reflect the Raw and/or the lung parenchymal tissue resistance. Therefore, in this study, the Penh is considered as a total lung resistance index (iRL), as has been previously proposed by others and us (1, 6, 12, 21). Moreover, in the present study, we used this plethysmographic technique to measure acute responses to increasing concentrations of ACh or His. These responses were characterized by a transient increase in iRL values, spontaneously returning to baseline levels after ~10 min, implying the reversibility of the RL increment, and thus pointing out that airway obstruction was the main component of such increment. This consideration is in agreement with the recent finding that airway sensitivity to His observed in guinea pigs through Penh measurement was very similar to the one obtained by specific Raw measurement (7). Therefore, it is highly feasible that, in our study, iRL mainly reflects Raw.
In this plethysmographic chamber, two routes of drug administration were used: inhalatory (SB/NEB group) and intravenous (SB/IV group). In the latter case, ACh or His was administered through a catheter placed in the left jugular vein, and every guinea pig was put into a metallic mesh that restricted its wide movements, limiting in this way the possibility that the catheter could be pulled out by the animal. Because of these last maneuvers (restriction of movements and invasive procedures), comparison with freely moving animals might not be appropriate. Therefore, an additional SB/NEB group was submitted to the same maneuvers as for SB/IV.
Plethysmography for mechanically ventilated animals. RL was measured through the isovolumetric method in a closed-chamber plethysmograph (Buxco Electronics). Guinea pigs were anesthetized with pentobarbital sodium (35 mg/kg ip), and the depth of anesthesia was kept with hourly administration of additional doses of pentobarbital (~9 mg/kg iv). Each animal received pancuronium bromide (0.06 mg/kg iv) to avoid spontaneous respiratory movements. After the trachea was cannulated, each animal was mechanically ventilated (model 50-1700, Harvard Apparatus) with a tidal volume of 10 ml/kg and 48 breaths/min. Right jugular vein and left carotid artery were cannulated for drug administration and for arterial pressure recording through a Beckman R-612 dynograph, respectively. A water-filled cannula was positioned into the middle one-third of the esophagus to measure intraesophageal pressure as a surrogate of intrapleural pressure. Pressures obtained from the esophageal and tracheal cannulas were recorded through a differential pressure transducer (SCXL004DN, Sen Sym). Pressure inside the plethysmograph chamber was also recorded through a differential pressure transducer. This last signal was converted to a pseudoflow signal through software (Buxco Biosystem XA, version 1.0). Finally, this software also calculated the relationship between both parameters to obtain RL through the formula RL = P/, where P is pressure change and is flow change.
Bronchial challenge. Airway responsiveness was assessed through bronchial challenges using inhaled or intravenous agonists in both spontaneously breathing and mechanically ventilated guinea pigs.
The nebulized bronchial challenge in spontaneously breathing animals was done in the plethysmographic chamber. Thus, after a baseline saline nebulization (0.9% NaCl, 2 min), noncumulative increasing doses of ACh (0.056-3.2 mg/ml) or His (0.018-3.2 mg/ml) were nebulized during 2 min each. The interval between doses was ~5-12 min, enough time to recover the basal iRL value. After each dose, the iRL was registered during 5 min, and the average value was calculated. The procedure ended when the iRL value after a certain dose was threefold or more of the basal iRL value (obtained after saline). Nebulizations were done by using a US-1 Bennett nebulizer (flow: 2 ml/min), with a mixed particle size of 44% < 4 μm, 38% = 4-10 μm, and 18% > 10 μm (multistage liquid impinger, 20 l/min, Bukard Manufacturing). In the case of mechanically ventilated guinea pigs, the nebulized agonists were delivered to the inlet port of the ventilator via a plastic reservoir, and in these last animals the exposure to nebulized agonist lasted only 1 min.
With regard to the intravenous bronchial challenge, after a baseline response to saline (0.1 ml iv of 0.9% NaCl) a dose-response curve for ACh (0.18-56 μg/kg iv) or His (0.18-56 μg/kg iv) was constructed. After each dose, the maximum response (iRL in spontaneously breathing guinea pigs, and RL in mechanically ventilated) obtained at any averaged 10-breath period during the following 3 min was registered. As described in the previous paragraph, the dose-response curve was finished when a response was threefold the baseline iRL or RL value.
O3 exposure. Animals were placed in a Plexiglas exposure chamber (48 × 73 × 32 cm) where they were continuously exposed to 3 parts/million (ppm) O3 during 1 h. O3 was produced by passing a constant air flow (3 l/min) through an ozonizer (Puraqua-V, Purificadores Eléctricos por Ozono, Mexico DF, Mexico), in which an electric arc decomposes air into O3. The O3 concentration inside the exposure chamber was regulated by modifying the voltage delivered to the ozonizer and monitored by an ultraviolet O3 analyzer (model 1008 PC, Dasibi Environmental).
Sensitization procedure and antigenic challenge. Guinea pigs were sensitized at day 0 by intraperitoneal administration of 40 μg ovalbumin and 1 mg Al(OH)3 in 0.5 ml of saline solution. On day 8, the animals were nebulized with 3 mg/ml ovalbumin in saline solution for 2 min, delivered by a US-Bennett nebulizer. On day 15, guinea pigs were nebulized again with 0.5 mg/ml ovalbumin in saline solution for 1 min. Animals were studied on days 21-25. Antigenic challenge was accomplished by delivering 0.5 mg/ml ovalbumin during 30 s by the inhalatory route.
AChE activity. The AChE activity was determined in guinea pig total lungs and blood samples by using a colorimetric method based on the Ellman reaction (9). Under deep anesthesia with an overdose of pentobarbital, the guinea pig's chest was opened, and a heparinized blood sample was obtained from cardiac puncture and refrigerated. Afterwards, lungs were washed by injecting phosphate buffer through the right ventricle, and all lung lobes were removed and stored at 20°C. On the next day, the lung tissue was homogenized in phosphate buffer (100 mg tissue/ml phosphate buffer) with a homogenizer (Polytron, Brinkmann Instruments, Westbury, NY). The homogenate was centrifuged for 15 min at 3,000 g, and the supernatant was filtered (22 μm polytetrafluoroethylene filter). Three hundred microliters of supernatant were added to a cuvette containing 2.5 ml of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; 0.32 mM) and 300 μl phosphate buffer (64 mM). The background absorbance per minute of each sample was measured at 405 nm at 25°C with a spectrophotometer (DU 640, Beckman, Fullerton, CA). Afterwards, 100 μl of AChE substrate (42 mM acetylthiocholine) were added to the cuvette, and the change in absorbance per minute was measured. Once the background absorbance was subtracted, the AChE activity was calculated as international units (IU) by means of the following equation
where A is the change in absorbance per minute; 1.36 × 104 is the extinction coefficient of DTNB; Co is the amount of tissue in the supernatant (mg tissue/ml buffer); 3,200 is the total volume (μl) of the cuvette; and 300 is the volume (μl) of the supernatant sample.
Regarding blood samples, after 1:25 dilution in phosphate buffer, 100 μl were added to a cuvette containing 2.5 ml of DTNB and 500 μl phosphate buffer. The remaining steps were the same as those described for lung homogenates, with the corresponding change in the formula (100 μl of sample instead of 300 μl).
Drugs. ACh chloride and His dihydrochloride (Sigma Chemical, St. Louis, MO) were dissolved in saline solution. Acetylthiocholine and DTNB were purchased from Aldrich Chemical (Milwaukee, WI) and were dissolved in Tris-phosphate buffer at pH 7.38.
Statistical analysis. For the assessment of airway responsiveness, the provocative agonist dose that caused a 200% increment in RL and iRL above baseline values (PD200) was calculated by interpolation in a straight-line regression analysis. Student's t-tests for unpaired data were used in most cases and for paired data in those animals receiving two dose-response curves sequentially. Statistical significance was set at two-tailed P < 0.05. Data are expressed in the text and in Fig. 7 as means ± SE.
RESULTS
Basal values of pulmonary function and their relationship with airway responsiveness. Table 1 shows the baseline values from every studied group, either as iRL or RL, according to the plethysmographic method used. There were no differences in most experimental vs. control pairwise comparisons, except in three pairs of groups. Additionally, there was no correlation between baseline iRL or RL and their corresponding PD200 value (data not shown).
SB/NEB (freely moving) group. When the inhalatory route was used to measure airway responsiveness through plethysmography for freely moving animals, the control group exposed to air showed lower responses to the second ACh curve, i.e., a clear hyporesponsiveness was developed (PD200: 0.41 ± 0.12 mg/ml, first curve vs. 0.86 ± 0.24 mg/ml, second curve; n = 6; P < 0.05; Fig. 1A). One-hour exposure to O3 did not modify the airway responsiveness to ACh (PD200: 0.50 ± 0.07 mg/ml before vs. 0.96 ± 0.24 mg/ml after O3; n = 6; Fig. 1B). However, when the duration of O3 exposure lasted longer (3 h), hyperresponsiveness to ACh was observed (PD200: 0.40 ± 0.06 mg/ml before vs. 0.27 ± 0.09 mg/ml after O3; n = 11; P < 0.05; Fig. 1C).
The PD200 to aerosolized His was not different before and after exposure to air (PD200: 0.048 ± 0.01 vs. 0.052 ± 0.01 mg/ml; n = 7; Fig. 2) or before and after O3 exposure for 1 h (0.06 ± 0.02 vs. 0.05 ± 0.01 mg/ml; n = 6).
Finally, airway hyperresponsiveness was developed in ovalbumin-sensitized animals after an antigenic challenge, as PD200 of nebulized ACh was statistically lower after such challenge (0.24 ± 0.10 vs. 0.67 ± 0.12 mg/ml; n = 5; P < 0.0005; Fig. 1D).
SB/NEB (restricted movements) group. O3 exposure did not modify the ACh and His airway responsiveness compared with their respective air-exposed groups. Thus similar PD200 values were observed after air and O3 exposure, either for the ACh challenge [0.68 ± 0.18 mg/ml (n = 6) vs. 0.56 ± 0.10 mg/ml (n = 4); P = 0.65; Fig. 3A] and the His challenge [0.07 ± 0.01 mg/ml (n = 4) vs. 0.07 ± 0.01 mg/ml (n = 4); P = 0.94; Fig. 4A].
SB/IV (restricted movements) group. Contrasting with the SB/NEB (restricted movements) group, when ACh or His were delivered intravenously, O3 generated airway hyperresponsiveness to both agonists with the shortest exposure (1 h) (Figs. 3B and 4B). Thus ACh or His PD200 values in guinea pigs submitted to O3 [7.36 ± 2.74 μg/kg (n = 6) and 1.41 ± 0.51 μg/kg (n = 6), respectively] were significantly lower than in those animals just receiving air [25.14 ± 4.28 μg/kg (n = 7), P < 0.01; and 9.38 ± 1.78 μg/kg (n = 7), P < 0.005, respectively].
MV/NEB group. O3 exposure did not change the airway responsiveness when nebulized agonists were administered to mechanically ventilated animals, as ACh PD200 values (0.84 ± 0.19 mg/ml; n = 6) were not statistically different from those observed in the air-exposed group (1.61 ± 0.30 mg/ml; n = 7; P = 0.062; Fig. 5A). Similarly, His PD200 values between O3- and air-exposed groups were similar [0.15 ± 0.06 mg/ml (n = 7) and 0.19 ± 0.07 mg/ml (n = 7), respectively; P = 0.70; Fig. 6A].
MV/IV group. By using anesthetized animals, exposure to 3 ppm O3 during 1 h generated airway hyperresponsiveness to the intravenous ACh and His. Thus after O3, ACh PD200 (10.26 ± 2.43 μg/kg; n = 6; Fig. 5B) was significantly lower compared with the air group (50.38 ± 16.14 μg/kg; n = 6; P < 0.05). Similarly, His PD200 was significantly lower for the O3-exposed group than for air-exposed animals [8.85 ± 1.94 μg/kg (n = 6) vs. 23.39 ± 5.50 μg/kg (n = 5), respectively; P < 0.02; Fig. 6B].
AChE activity. O3 exposure did not cause statistical changes in the AChE activity in either lung homogenates [1.022 ± 0.345 IU (n = 6) vs. control, 0.301 ± 0.069 IU (n = 5); P = 0.09] or total blood samples [1.926 ± 0.470 IU (n = 6) vs. control, 1.320 ± 0.232 IU (n = 4); P = 0.35; Fig. 7].
DISCUSSION
O3-induced airway hyperresponsiveness is a well-characterized phenomenon widely studied in laboratory animals since many years ago. Nevertheless, several factors might influence the resulting effects of O3, such as concentration and duration of the exposure, time elapsed between exposure and bronchoprovocation tests, and nature of the bronchoconstrictor agent used to demonstrate the hyperresponsiveness. An additional factor that has been very scantily studied is the administration route utilized to deliver the bronchoconstrictor agonist (18). In the present study, we found that, at least under our experimental conditions (3 ppm O3 during 1 h), bronchoprovocation tests performed with nebulized agents were unable to demonstrate the O3-induced airway hyperresponsiveness (as can be seen in Figs. 1B, 2, 3A, 4A, 5A, and 6A), whereas intravenously delivered agents always revealed it (as observed in Figs. 3B, 4B, 5B, and 6B). These contrasting responses were independent of the plethysmographic method used.
Our results differ from some previously published works made in guinea pigs, in which airway hyperresponsiveness to inhaled agonist was found shortly after a 3 ppm O3 exposure during 1 h or less (10, 14, 17, 23). Most of these studies, however, measured the airway reactivity when a significant change in baseline airway caliber was present (between 60 and 120% increment), and thus such hyperresponsiveness could be explained solely due to mechanical reasons. In this sense, we have found that this transient bronchoconstriction might last for up to 3 h (21), and thus O3-induced airway hyperresponsiveness due to nonmechanical processes should be measured once this obstructing phase has ended. Although we took care to avoid large O3-induced changes in the basal values of iRL and RL, we found a statistically significant increase in two groups. In both cases, the increment was <19%, and an associated hyperresponsiveness was observed in only one of them. Moreover, there was no correlation between iRL or RL basal values and their corresponding airway responsiveness, either in individual groups or in pooled SB/NEB freely moving animals (data not shown). Thus it is probable that the O3-induced changes in airway responsiveness obtained in our study were not due to modifications in basal values.
Contrasting with our results observed with 3 ppm O3 exposure during 1 h, we observed that a longer exposure time (3 h) was capable of producing airway hyperresponsiveness to inhaled agonists (Fig. 1, B and C, respectively). In this sense, some researchers have exposed guinea pigs to the same O3 concentration for up to 2 h and found airway hyperresponsiveness at 5 h (17), or at 4, 14, and 24 h (18) after the exposure. Therefore, at least at 3 ppm, duration of the exposure to O3 seems to influence the development of airway hyperresponsiveness to inhaled agonists.
In general, our results strongly suggest that the administration route was the main factor responsible for the characterization of the O3-induced airway hyperresponsiveness. This is in agreement with a previous report by Roum and Murlas (18), in which they concluded that the inhalatory technique was not appropriate to demonstrate O3-induced airway hyperresponsiveness to cholinergic agonist because of its great variability, whereas the intravenous method was more consistent. The disparate responses observed when bronchoconstrictors were delivered via aerosols or intravenously might have several explanations.
First, previous studies have shown that O3 exposure causes morphological (4, 13, 19) and functional (3) changes in the peripheral airways. Thus, if peripheral airways are the site at which O3 is causing the major functional disturbances, one possibility is that inhaled bronchoconstrictor agonists did not properly reach such distal regions. The intravenous route, by contrast, should reach all of these locations homogeneously, thus detecting the O3-induced dysfunction. A different site of action of inhaled and intravenous agonists can also be inferred from the study by Corddry et al. (8). They found that responsiveness measured through whole RL and peripheral Raw closely matched during an intravenous bronchial challenge to His, whereas both measurements yielded different results with nebulized His. This argument might also be reasonable to interpret the results obtained by increasing the time of O3 exposure to 3 h (Fig. 1C), because this stronger stimulus could be producing dysfunction at a more proximal airway level. This postulate would also explain the airway hyperresponsiveness to aerosolized agents after antigen challenge in sensitized guinea pigs observed by others (5) and us (Fig. 1D), because it is well known that sensitization in animal models occurs at all airway levels.
Second, an increased arrival of the intravenously administered agonists to the airway smooth muscle would also explain the different effect of O3 on airway hyperresponsiveness. In this sense, it is known that O3 exposure produces an increased vascular permeability (16), which might favor an augmented delivery of the agonists to the airway smooth muscle that would not occur when the agonists are delivered by the inhalatory route. Further research is needed to assess the certainty of this hypothesis. On the other hand, some studies have found that tissue and blood AChE activity diminishes shortly after O3 exposure (2, 11). Thus it is reasonable to speculate that, in our study, the inhibition of AChE in lung tissue was not enough to produce airway hyperresponsiveness to inhaled ACh, but, when the intravenous route was used, the additional AChE inhibition in blood would cause more ACh to reach the airway smooth muscle, thus causing airway hyperresponsiveness. However, we found that, under our experimental conditions, O3 did not inhibit AChE activity (in fact, an opposite trend was noticed; Fig. 7). This discrepancy with published works might be explained by the longer lapse between the O3 exposure and the AChE activity measurement in our work. In fact, in preliminary experiments, we have corroborated that an acute O3 exposure (3 ppm, 1 h) causes a transient diminution of blood AChE activity in rabbits, which returned to control levels at 16-18 h after exposure (data not shown). Thus AChE inhibition seems not to be involved in the differential effects of inhaled and intravenous agonists to demonstrate O3-induced airway hyperresponsiveness.
Finally, as can be seen in Fig. 1A, in the air-exposed SB/NEB freely moving animals, we found that a second administration of aerosolized ACh was notably less efficacious than the first one, i.e., a hyporesponsiveness to this agonist was developed. This phenomenon did not occur when His was nebulized nor when either agonist was administered by the intravenous route (data not shown). The nature of this ACh-induced hyporesponsiveness is unclear. It might be possible that aerosolized ACh activated prejunctional M2 receptors (with a consequent inhibition of basal ACh release) or promoted the production of a relaxant factor more effectively than intravenous ACh. Nonetheless, the role of this phenomenon, if any, in the lack of O3-induced hyperresponsiveness to inhaled agonists remains speculative and deserves further investigation.
In conclusion, in the two plethysmographic methods used by us, we found that inhaled agonists were less effective to reveal the airway hyperresponsiveness after an acute O3 exposure than intravenous ones, at least for the 1-h exposure to 3 ppm. This difference seems not to be related to an O3-induced inhibition of the AChE activity.
ACKNOWLEDGEMENTS
Consejo Nacional de Ciencia y Tecnologia and Instituto Mexicano del Seguro Social provided scholarships supporting postgraduate studies in which this work was done (to B. Sommer).
FOOTNOTES
* L. M. Monta?o and M. H. Vargas contributed equally to this work.
Address for reprint requests and other correspondence: L. M. Monta?o, Departamento de Investigación en Asma, Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502, CP 14080, México DF, México (E-mail: lmmr@servidor.unam.mx).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 December 2000; accepted in final form 15 August 2001.
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ABSTRACT
Ozone (O3)-induced airway hyperresponsiveness in laboratory animals is usually demonstrated through dose-response curves with inhaled or intravenous bronchoconstrictor agonists. However, comparability of these two routes has not been well documented. Thus guinea pig airway responsiveness to ACh and histamine was evaluated 16-18 h after O3 (3 parts/million, 1 h) or air exposure by two plethysmographic methods (spontaneously breathing and mechanically ventilated) and by two administration routes (inhalatory or intravenous). We found that O3 caused airway hyperresponsiveness to intravenous, but not to inhaled, agonists, independent of the plethysmographic method used. Suitability of the inhalatory route to detect airway hyperresponsiveness was corroborated with inhaled ACh after an antigen challenge or extending O3 exposure to 3 h. Acetylcholinesterase activity was not modified after O3 exposure in lung homogenates and blood samples. Thus inhaled agonists were less effective to reveal the airway hyperresponsiveness after an acute O3 exposure than intravenous ones, at least for the 1-h exposure to 3 parts/million, and this difference seems not to be related to an O3-induced inhibition of the acetylcholinesterase activity.
keywords:ozone; airway hyperresponsiveness; dose-response curve; guinea pig
INTRODUCTION
EXPOSURE TO OZONE (O3) is a widely used method to induce airway hyperresponsiveness in different species, including guinea pigs and humans (3, 15, 20). The increased responsiveness is usually demonstrated through pharmacological methods, i.e., the elaboration of dose-response curves with bronchoconstrictor agents. In laboratory animals, the most common ways to deliver these agents to the airways are the intravenous (22, 23) and the inhalatory routes (14, 17). Although extensively used to demonstrate this phenomenon, differences between inhaled and intravenously administered agents have been very scantily investigated (18). This is an important issue because conclusions derived from experiments using one route might not be comparable with those using the other one, unless equivalence of both routes was assessed. In this sense, Corddry et al. (8) have demonstrated in dogs that whole lung resistance (RL) and peripheral airway resistance (Raw) measurements detected the same pattern of responsiveness to intravenous histamine (His), whereas they yielded different results with nebulized His. In the present work, we found that, at a certain O3 exposure magnitude, inhaled agonists did not detect airway hyperresponsiveness as the intravenous ones did, and some possible mechanisms for this difference are discussed.
MATERIALS AND METHODS
Animals and experimental design. Male Hartley guinea pigs (450-500 g body wt), bred in conventional conditions (filtered conditioned air, 21 ± 1°C, 50-70% humidity, sterilized bed) and fed with Purina pellets supplemented with disinfected fresh alfalfa and sterilized water, were used. Airway hyperresponsiveness to ACh and His was assessed by constructing dose-response curves. Two methods were used to measure the responses: plethysmography in spontaneously breathing (SB) animals and plethysmography in mechanically ventilated (MV) animals. In either method, bronchial challenges were accomplished by delivering the drugs by nebulized (NEB) or intravenous (IV) route. Therefore, four main groups of animals were integrated: SB/NEB challenge, SB/IV challenge, MV/NEB challenge, and MV/IV challenge.
With regard to the SB/NEB group, the noninvasive nature of the technique allowed us to perform two doses-response curves in the same guinea pigs. In these animals, the first curve was done ~24 h before the O3 or air (control group) exposure, and the second was done 16-18 h after such exposure. In the remaining groups, only one dose-response curve after air or O3 exposure was performed in each animal. In addition, a group of sensitized guinea pigs was submitted to a dose-response curve to inhaled ACh 24 h before and 3 h after an antigenic challenge. Finally, separate groups of guinea pigs with or without O3 exposure were used to evaluate the acetylcholinesterase (AChE) activity in lung homogenates and blood.
Plethysmography in spontaneously breathing animals. Each guinea pig was introduced into a plethysmographic chamber designed for unrestrained, freely moving animals (Buxco Electronics, Troy, NY). This chamber is supplied with a constant air flow (10 ml/s) that does not alter the respiratory signals. This methodology has been comprehensively described elsewhere (5, 6, 12). Briefly, the pressure inside the chamber was measured by a differential pressure transducer (SCXL004DN, Sen Sym, Milpitas, CA) connected to a preamplifier and continuously monitored through software (Buxco Biosystem XA, version 1.1). Changes in box pressure represent the difference between the thoracic expansion or contraction and the tidal volume (air removed from or added to the chamber during inspiration or expiration). The box pressure is differentiated to give a pseudoflow signal, which is then analyzed by the software to give an index named "enhanced pause" (Penh). The Penh value is obtained for each respiration by the following formula
where TE is expiratory time, RT is relaxation time, PEpeak is peak expiratory pressure, and PIpeak is peak inspiratory pressure. Penh values used in our study were obtained from averaging logged values every 15 s. The software was adjusted to only include breaths with a tidal value of 1 ml, with a minimal inspiratory time of 0.15 s, a maximal inspiratory time of 3 s, and a maximal difference between inspiratory and expiratory volumes of 10%. A recent study in mice (12) demonstrated that Penh had a close, positive correlation with the measurement of total RL, which in turn can reflect the Raw and/or the lung parenchymal tissue resistance. Therefore, in this study, the Penh is considered as a total lung resistance index (iRL), as has been previously proposed by others and us (1, 6, 12, 21). Moreover, in the present study, we used this plethysmographic technique to measure acute responses to increasing concentrations of ACh or His. These responses were characterized by a transient increase in iRL values, spontaneously returning to baseline levels after ~10 min, implying the reversibility of the RL increment, and thus pointing out that airway obstruction was the main component of such increment. This consideration is in agreement with the recent finding that airway sensitivity to His observed in guinea pigs through Penh measurement was very similar to the one obtained by specific Raw measurement (7). Therefore, it is highly feasible that, in our study, iRL mainly reflects Raw.
In this plethysmographic chamber, two routes of drug administration were used: inhalatory (SB/NEB group) and intravenous (SB/IV group). In the latter case, ACh or His was administered through a catheter placed in the left jugular vein, and every guinea pig was put into a metallic mesh that restricted its wide movements, limiting in this way the possibility that the catheter could be pulled out by the animal. Because of these last maneuvers (restriction of movements and invasive procedures), comparison with freely moving animals might not be appropriate. Therefore, an additional SB/NEB group was submitted to the same maneuvers as for SB/IV.
Plethysmography for mechanically ventilated animals. RL was measured through the isovolumetric method in a closed-chamber plethysmograph (Buxco Electronics). Guinea pigs were anesthetized with pentobarbital sodium (35 mg/kg ip), and the depth of anesthesia was kept with hourly administration of additional doses of pentobarbital (~9 mg/kg iv). Each animal received pancuronium bromide (0.06 mg/kg iv) to avoid spontaneous respiratory movements. After the trachea was cannulated, each animal was mechanically ventilated (model 50-1700, Harvard Apparatus) with a tidal volume of 10 ml/kg and 48 breaths/min. Right jugular vein and left carotid artery were cannulated for drug administration and for arterial pressure recording through a Beckman R-612 dynograph, respectively. A water-filled cannula was positioned into the middle one-third of the esophagus to measure intraesophageal pressure as a surrogate of intrapleural pressure. Pressures obtained from the esophageal and tracheal cannulas were recorded through a differential pressure transducer (SCXL004DN, Sen Sym). Pressure inside the plethysmograph chamber was also recorded through a differential pressure transducer. This last signal was converted to a pseudoflow signal through software (Buxco Biosystem XA, version 1.0). Finally, this software also calculated the relationship between both parameters to obtain RL through the formula RL = P/, where P is pressure change and is flow change.
Bronchial challenge. Airway responsiveness was assessed through bronchial challenges using inhaled or intravenous agonists in both spontaneously breathing and mechanically ventilated guinea pigs.
The nebulized bronchial challenge in spontaneously breathing animals was done in the plethysmographic chamber. Thus, after a baseline saline nebulization (0.9% NaCl, 2 min), noncumulative increasing doses of ACh (0.056-3.2 mg/ml) or His (0.018-3.2 mg/ml) were nebulized during 2 min each. The interval between doses was ~5-12 min, enough time to recover the basal iRL value. After each dose, the iRL was registered during 5 min, and the average value was calculated. The procedure ended when the iRL value after a certain dose was threefold or more of the basal iRL value (obtained after saline). Nebulizations were done by using a US-1 Bennett nebulizer (flow: 2 ml/min), with a mixed particle size of 44% < 4 μm, 38% = 4-10 μm, and 18% > 10 μm (multistage liquid impinger, 20 l/min, Bukard Manufacturing). In the case of mechanically ventilated guinea pigs, the nebulized agonists were delivered to the inlet port of the ventilator via a plastic reservoir, and in these last animals the exposure to nebulized agonist lasted only 1 min.
With regard to the intravenous bronchial challenge, after a baseline response to saline (0.1 ml iv of 0.9% NaCl) a dose-response curve for ACh (0.18-56 μg/kg iv) or His (0.18-56 μg/kg iv) was constructed. After each dose, the maximum response (iRL in spontaneously breathing guinea pigs, and RL in mechanically ventilated) obtained at any averaged 10-breath period during the following 3 min was registered. As described in the previous paragraph, the dose-response curve was finished when a response was threefold the baseline iRL or RL value.
O3 exposure. Animals were placed in a Plexiglas exposure chamber (48 × 73 × 32 cm) where they were continuously exposed to 3 parts/million (ppm) O3 during 1 h. O3 was produced by passing a constant air flow (3 l/min) through an ozonizer (Puraqua-V, Purificadores Eléctricos por Ozono, Mexico DF, Mexico), in which an electric arc decomposes air into O3. The O3 concentration inside the exposure chamber was regulated by modifying the voltage delivered to the ozonizer and monitored by an ultraviolet O3 analyzer (model 1008 PC, Dasibi Environmental).
Sensitization procedure and antigenic challenge. Guinea pigs were sensitized at day 0 by intraperitoneal administration of 40 μg ovalbumin and 1 mg Al(OH)3 in 0.5 ml of saline solution. On day 8, the animals were nebulized with 3 mg/ml ovalbumin in saline solution for 2 min, delivered by a US-Bennett nebulizer. On day 15, guinea pigs were nebulized again with 0.5 mg/ml ovalbumin in saline solution for 1 min. Animals were studied on days 21-25. Antigenic challenge was accomplished by delivering 0.5 mg/ml ovalbumin during 30 s by the inhalatory route.
AChE activity. The AChE activity was determined in guinea pig total lungs and blood samples by using a colorimetric method based on the Ellman reaction (9). Under deep anesthesia with an overdose of pentobarbital, the guinea pig's chest was opened, and a heparinized blood sample was obtained from cardiac puncture and refrigerated. Afterwards, lungs were washed by injecting phosphate buffer through the right ventricle, and all lung lobes were removed and stored at 20°C. On the next day, the lung tissue was homogenized in phosphate buffer (100 mg tissue/ml phosphate buffer) with a homogenizer (Polytron, Brinkmann Instruments, Westbury, NY). The homogenate was centrifuged for 15 min at 3,000 g, and the supernatant was filtered (22 μm polytetrafluoroethylene filter). Three hundred microliters of supernatant were added to a cuvette containing 2.5 ml of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; 0.32 mM) and 300 μl phosphate buffer (64 mM). The background absorbance per minute of each sample was measured at 405 nm at 25°C with a spectrophotometer (DU 640, Beckman, Fullerton, CA). Afterwards, 100 μl of AChE substrate (42 mM acetylthiocholine) were added to the cuvette, and the change in absorbance per minute was measured. Once the background absorbance was subtracted, the AChE activity was calculated as international units (IU) by means of the following equation
where A is the change in absorbance per minute; 1.36 × 104 is the extinction coefficient of DTNB; Co is the amount of tissue in the supernatant (mg tissue/ml buffer); 3,200 is the total volume (μl) of the cuvette; and 300 is the volume (μl) of the supernatant sample.
Regarding blood samples, after 1:25 dilution in phosphate buffer, 100 μl were added to a cuvette containing 2.5 ml of DTNB and 500 μl phosphate buffer. The remaining steps were the same as those described for lung homogenates, with the corresponding change in the formula (100 μl of sample instead of 300 μl).
Drugs. ACh chloride and His dihydrochloride (Sigma Chemical, St. Louis, MO) were dissolved in saline solution. Acetylthiocholine and DTNB were purchased from Aldrich Chemical (Milwaukee, WI) and were dissolved in Tris-phosphate buffer at pH 7.38.
Statistical analysis. For the assessment of airway responsiveness, the provocative agonist dose that caused a 200% increment in RL and iRL above baseline values (PD200) was calculated by interpolation in a straight-line regression analysis. Student's t-tests for unpaired data were used in most cases and for paired data in those animals receiving two dose-response curves sequentially. Statistical significance was set at two-tailed P < 0.05. Data are expressed in the text and in Fig. 7 as means ± SE.
RESULTS
Basal values of pulmonary function and their relationship with airway responsiveness. Table 1 shows the baseline values from every studied group, either as iRL or RL, according to the plethysmographic method used. There were no differences in most experimental vs. control pairwise comparisons, except in three pairs of groups. Additionally, there was no correlation between baseline iRL or RL and their corresponding PD200 value (data not shown).
SB/NEB (freely moving) group. When the inhalatory route was used to measure airway responsiveness through plethysmography for freely moving animals, the control group exposed to air showed lower responses to the second ACh curve, i.e., a clear hyporesponsiveness was developed (PD200: 0.41 ± 0.12 mg/ml, first curve vs. 0.86 ± 0.24 mg/ml, second curve; n = 6; P < 0.05; Fig. 1A). One-hour exposure to O3 did not modify the airway responsiveness to ACh (PD200: 0.50 ± 0.07 mg/ml before vs. 0.96 ± 0.24 mg/ml after O3; n = 6; Fig. 1B). However, when the duration of O3 exposure lasted longer (3 h), hyperresponsiveness to ACh was observed (PD200: 0.40 ± 0.06 mg/ml before vs. 0.27 ± 0.09 mg/ml after O3; n = 11; P < 0.05; Fig. 1C).
The PD200 to aerosolized His was not different before and after exposure to air (PD200: 0.048 ± 0.01 vs. 0.052 ± 0.01 mg/ml; n = 7; Fig. 2) or before and after O3 exposure for 1 h (0.06 ± 0.02 vs. 0.05 ± 0.01 mg/ml; n = 6).
Finally, airway hyperresponsiveness was developed in ovalbumin-sensitized animals after an antigenic challenge, as PD200 of nebulized ACh was statistically lower after such challenge (0.24 ± 0.10 vs. 0.67 ± 0.12 mg/ml; n = 5; P < 0.0005; Fig. 1D).
SB/NEB (restricted movements) group. O3 exposure did not modify the ACh and His airway responsiveness compared with their respective air-exposed groups. Thus similar PD200 values were observed after air and O3 exposure, either for the ACh challenge [0.68 ± 0.18 mg/ml (n = 6) vs. 0.56 ± 0.10 mg/ml (n = 4); P = 0.65; Fig. 3A] and the His challenge [0.07 ± 0.01 mg/ml (n = 4) vs. 0.07 ± 0.01 mg/ml (n = 4); P = 0.94; Fig. 4A].
SB/IV (restricted movements) group. Contrasting with the SB/NEB (restricted movements) group, when ACh or His were delivered intravenously, O3 generated airway hyperresponsiveness to both agonists with the shortest exposure (1 h) (Figs. 3B and 4B). Thus ACh or His PD200 values in guinea pigs submitted to O3 [7.36 ± 2.74 μg/kg (n = 6) and 1.41 ± 0.51 μg/kg (n = 6), respectively] were significantly lower than in those animals just receiving air [25.14 ± 4.28 μg/kg (n = 7), P < 0.01; and 9.38 ± 1.78 μg/kg (n = 7), P < 0.005, respectively].
MV/NEB group. O3 exposure did not change the airway responsiveness when nebulized agonists were administered to mechanically ventilated animals, as ACh PD200 values (0.84 ± 0.19 mg/ml; n = 6) were not statistically different from those observed in the air-exposed group (1.61 ± 0.30 mg/ml; n = 7; P = 0.062; Fig. 5A). Similarly, His PD200 values between O3- and air-exposed groups were similar [0.15 ± 0.06 mg/ml (n = 7) and 0.19 ± 0.07 mg/ml (n = 7), respectively; P = 0.70; Fig. 6A].
MV/IV group. By using anesthetized animals, exposure to 3 ppm O3 during 1 h generated airway hyperresponsiveness to the intravenous ACh and His. Thus after O3, ACh PD200 (10.26 ± 2.43 μg/kg; n = 6; Fig. 5B) was significantly lower compared with the air group (50.38 ± 16.14 μg/kg; n = 6; P < 0.05). Similarly, His PD200 was significantly lower for the O3-exposed group than for air-exposed animals [8.85 ± 1.94 μg/kg (n = 6) vs. 23.39 ± 5.50 μg/kg (n = 5), respectively; P < 0.02; Fig. 6B].
AChE activity. O3 exposure did not cause statistical changes in the AChE activity in either lung homogenates [1.022 ± 0.345 IU (n = 6) vs. control, 0.301 ± 0.069 IU (n = 5); P = 0.09] or total blood samples [1.926 ± 0.470 IU (n = 6) vs. control, 1.320 ± 0.232 IU (n = 4); P = 0.35; Fig. 7].
DISCUSSION
O3-induced airway hyperresponsiveness is a well-characterized phenomenon widely studied in laboratory animals since many years ago. Nevertheless, several factors might influence the resulting effects of O3, such as concentration and duration of the exposure, time elapsed between exposure and bronchoprovocation tests, and nature of the bronchoconstrictor agent used to demonstrate the hyperresponsiveness. An additional factor that has been very scantily studied is the administration route utilized to deliver the bronchoconstrictor agonist (18). In the present study, we found that, at least under our experimental conditions (3 ppm O3 during 1 h), bronchoprovocation tests performed with nebulized agents were unable to demonstrate the O3-induced airway hyperresponsiveness (as can be seen in Figs. 1B, 2, 3A, 4A, 5A, and 6A), whereas intravenously delivered agents always revealed it (as observed in Figs. 3B, 4B, 5B, and 6B). These contrasting responses were independent of the plethysmographic method used.
Our results differ from some previously published works made in guinea pigs, in which airway hyperresponsiveness to inhaled agonist was found shortly after a 3 ppm O3 exposure during 1 h or less (10, 14, 17, 23). Most of these studies, however, measured the airway reactivity when a significant change in baseline airway caliber was present (between 60 and 120% increment), and thus such hyperresponsiveness could be explained solely due to mechanical reasons. In this sense, we have found that this transient bronchoconstriction might last for up to 3 h (21), and thus O3-induced airway hyperresponsiveness due to nonmechanical processes should be measured once this obstructing phase has ended. Although we took care to avoid large O3-induced changes in the basal values of iRL and RL, we found a statistically significant increase in two groups. In both cases, the increment was <19%, and an associated hyperresponsiveness was observed in only one of them. Moreover, there was no correlation between iRL or RL basal values and their corresponding airway responsiveness, either in individual groups or in pooled SB/NEB freely moving animals (data not shown). Thus it is probable that the O3-induced changes in airway responsiveness obtained in our study were not due to modifications in basal values.
Contrasting with our results observed with 3 ppm O3 exposure during 1 h, we observed that a longer exposure time (3 h) was capable of producing airway hyperresponsiveness to inhaled agonists (Fig. 1, B and C, respectively). In this sense, some researchers have exposed guinea pigs to the same O3 concentration for up to 2 h and found airway hyperresponsiveness at 5 h (17), or at 4, 14, and 24 h (18) after the exposure. Therefore, at least at 3 ppm, duration of the exposure to O3 seems to influence the development of airway hyperresponsiveness to inhaled agonists.
In general, our results strongly suggest that the administration route was the main factor responsible for the characterization of the O3-induced airway hyperresponsiveness. This is in agreement with a previous report by Roum and Murlas (18), in which they concluded that the inhalatory technique was not appropriate to demonstrate O3-induced airway hyperresponsiveness to cholinergic agonist because of its great variability, whereas the intravenous method was more consistent. The disparate responses observed when bronchoconstrictors were delivered via aerosols or intravenously might have several explanations.
First, previous studies have shown that O3 exposure causes morphological (4, 13, 19) and functional (3) changes in the peripheral airways. Thus, if peripheral airways are the site at which O3 is causing the major functional disturbances, one possibility is that inhaled bronchoconstrictor agonists did not properly reach such distal regions. The intravenous route, by contrast, should reach all of these locations homogeneously, thus detecting the O3-induced dysfunction. A different site of action of inhaled and intravenous agonists can also be inferred from the study by Corddry et al. (8). They found that responsiveness measured through whole RL and peripheral Raw closely matched during an intravenous bronchial challenge to His, whereas both measurements yielded different results with nebulized His. This argument might also be reasonable to interpret the results obtained by increasing the time of O3 exposure to 3 h (Fig. 1C), because this stronger stimulus could be producing dysfunction at a more proximal airway level. This postulate would also explain the airway hyperresponsiveness to aerosolized agents after antigen challenge in sensitized guinea pigs observed by others (5) and us (Fig. 1D), because it is well known that sensitization in animal models occurs at all airway levels.
Second, an increased arrival of the intravenously administered agonists to the airway smooth muscle would also explain the different effect of O3 on airway hyperresponsiveness. In this sense, it is known that O3 exposure produces an increased vascular permeability (16), which might favor an augmented delivery of the agonists to the airway smooth muscle that would not occur when the agonists are delivered by the inhalatory route. Further research is needed to assess the certainty of this hypothesis. On the other hand, some studies have found that tissue and blood AChE activity diminishes shortly after O3 exposure (2, 11). Thus it is reasonable to speculate that, in our study, the inhibition of AChE in lung tissue was not enough to produce airway hyperresponsiveness to inhaled ACh, but, when the intravenous route was used, the additional AChE inhibition in blood would cause more ACh to reach the airway smooth muscle, thus causing airway hyperresponsiveness. However, we found that, under our experimental conditions, O3 did not inhibit AChE activity (in fact, an opposite trend was noticed; Fig. 7). This discrepancy with published works might be explained by the longer lapse between the O3 exposure and the AChE activity measurement in our work. In fact, in preliminary experiments, we have corroborated that an acute O3 exposure (3 ppm, 1 h) causes a transient diminution of blood AChE activity in rabbits, which returned to control levels at 16-18 h after exposure (data not shown). Thus AChE inhibition seems not to be involved in the differential effects of inhaled and intravenous agonists to demonstrate O3-induced airway hyperresponsiveness.
Finally, as can be seen in Fig. 1A, in the air-exposed SB/NEB freely moving animals, we found that a second administration of aerosolized ACh was notably less efficacious than the first one, i.e., a hyporesponsiveness to this agonist was developed. This phenomenon did not occur when His was nebulized nor when either agonist was administered by the intravenous route (data not shown). The nature of this ACh-induced hyporesponsiveness is unclear. It might be possible that aerosolized ACh activated prejunctional M2 receptors (with a consequent inhibition of basal ACh release) or promoted the production of a relaxant factor more effectively than intravenous ACh. Nonetheless, the role of this phenomenon, if any, in the lack of O3-induced hyperresponsiveness to inhaled agonists remains speculative and deserves further investigation.
In conclusion, in the two plethysmographic methods used by us, we found that inhaled agonists were less effective to reveal the airway hyperresponsiveness after an acute O3 exposure than intravenous ones, at least for the 1-h exposure to 3 ppm. This difference seems not to be related to an O3-induced inhibition of the AChE activity.
ACKNOWLEDGEMENTS
Consejo Nacional de Ciencia y Tecnologia and Instituto Mexicano del Seguro Social provided scholarships supporting postgraduate studies in which this work was done (to B. Sommer).
FOOTNOTES
* L. M. Monta?o and M. H. Vargas contributed equally to this work.
Address for reprint requests and other correspondence: L. M. Monta?o, Departamento de Investigación en Asma, Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502, CP 14080, México DF, México (E-mail: lmmr@servidor.unam.mx).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 December 2000; accepted in final form 15 August 2001.
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