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Characterization of nasal obstruction in the allergic guinea pig using the forced oscillation method
Journal of Pharmacological and Toxicological Methods 48 (2002) 153 – 159 www.elsevier.com/locate/jpharmtox
Original article
Characterization of nasal obstruction in the allergic guinea pig using the forced oscillation method Robbie L. McLeoda,*, Simon S. Youngb, Christine H. Ericksona, Leonard E. Parraa, John A. Heya a
Department of Allergy, Schering-Plough Research Institute, 2015 Galloping Hill Road Kenilworth, NJ 07033-0539, USA b Discovery Bioscience, AstraZeneca, Bakewell Road, Loughborough, Leicestershire LE11 5RH, UK Received 13 March 2002; accepted 20 June 2003
Abstract Introduction: This is the first report to evaluate changes in nasal resistance in a preclinical animal model using the forced oscillation method. Methods: The method involves characterizing pressure – flow relationships of the respiratory system due to external oscillatory forces. Results: First, we evaluated changes in nasal resistance using an established small-animal rhinometric technique. In these studies, aerosolized ovalbumin (3%) administered to the nasal cavity of ovalbumin-sensitized guinea pigs increased nasal resistance at 30 min by 99 F 14%. The histamine H1 antagonists, chlorpheniramine (1 mg/kg iv) and pyrilamine (1 mg/kg iv), blocked the increase in nasal resistance due to ovalbumin provocation (50 F 17% and 39 F 11% over baseline, respectively). The a-adrenergic agonist phenylpropanolamine (3 mg/ kg iv) had no effect on the nasal actions of ovalbumin. In separate studies, nasal resistance was measured at 2 Hz by forced oscillation and ovalbumin (3%) increased nasal resistance by 91 F 14%. Chlorpheniramine (1 mg/kg iv) significantly attenuated the increase in nasal resistance due to ovalbumin. Finally, changes in nasal resistance for each treatment group were evaluated at frequencies of 1 – 18 Hz. Area under the curve analysis demonstrated that chlorpheniramine blocked the nasal obstructive effect of ovalbumin. In contrast, a pharmacologically active dose of phenylpropanolamine (3 mg/kg iv) did not produce decongestant activity. Discussion: The current data are inconsistent with the well-established clinical efficacy of a-adrenergic agonists as nasal decongestants. Consequently, we suggest that allergic nasal obstruction in the guinea pig may not be the best preclinical approach to assess the nasal decongestant activity of vasoconstrictor aadrenergic agonists. Additionally, our studies demonstrate the utility of the forced oscillation technique in assessing changes in nasal resistance in small laboratory animals. D 2003 Elsevier Inc. All rights reserved. Keywords: Forced oscillation; Guinea pig; Nasal congestion; Nasal obstruction; Nasal resistance; Phenylpropanolamine
1. Introduction The ability to reliably evaluate changes in nasal cavity patency in animals is of paramount importance in the preclinical evaluation of novel treatments for upper respiratory diseases, such as allergic rhinitis. The use of small animals, such as rat (Albert et al., 1998; Lau, King, & Boura, 1990), guinea pig (Narita & Asakura, 1993), rabbit (Benazzo et al., 1994; Bende, Hansell, Intaglietta, & ¨ nggard & Densert, 1974; Eccles & Arfors, 1992), cat (A Wilson, 1974; Malm, 1973; McLeod, Mingo, Herczku, DeGennaro-Culver, et al., 1999; McLeod, Mingo, Herczku,
* Corresponding author. Tel.: +1-908 740-3286; fax: +1-908-7407175. E-mail address: [email protected] (R.L. McLeod). 1056-8719/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1056-8719(03)00044-3
Corboz, et al., 1999), and large animals, such as dog (Dylewska, Sahin, & Widdicombe, 1993; Lacroix, Ulman, & Potter, 1994; Lung, Phipps, Wang, & Widdicombe, 1984) and pig (Eccles, 1978; Lacroix, 1989), have been used to generate important information on nasal physiology and pathophysiology. Moreover, many of these models have been beneficial in assessing the pharmacology of drugs used in the treatment of nasal diseases (Mizuno, Kawamura, Iwase, & Ohno, 1991). Current techniques used to study changes in nasal patency fall into one of two categories (Cole, Roithmann, Roth, & Chapnik, 1997). Dynamic techniques derive nasal resistance from measurements of nasal airflow and differential pressures between proximal and distal points of the nasal cavity. Static methods measure nasal cavity geometry independent of airflow. Both dynamic rhinomanometric methods that evaluate nasal airway resistance and static procedures, such as
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acoustic rhinometry are, approaches that have been employed to study nasal patency in animals (Erickson et al., 2001; McLeod, Mingo, Herczku, DeGennaro-Culver, et al., 1999; McLeod, Mingo, Herczku, Corboz, et al., 1999; Koss, Yu, Hey, & McLeod, 2002a,b; Salem & Clemente, 1972). Additionally, there are a few methods especially suited to study changes in nasal resistances in small laboratory animals, such as guinea pigs. However, none of these methods are ideal. For example, plethysmographic methods measure total respiratory resistance (pulmonary and nasal). Consequently, using this method, it is difficult to characterize changes in resistances due solely to changes in nasal patency. Therefore, characterizing new and potentially improved methods for evaluating nasal resistance, such as the forced oscillation technique, may be important for preclinical research and the development of novel therapies. The forced oscillation method was first introduced by Dubois, Brody, Lewis, and Burgess (1956) to study pulmonary mechanics. The technique involves characterizing the pressure – flow relationship of the respiratory system due to external oscillatory forces. Estimates of pulmonary resistance and reactance are determined as a function of frequency (Watson, Jackson, & Drazen, 1986). A few clinical investigators have applied the forced oscillation method to assess nasal resistance in humans (Aksamit, Duggan, Watson, & Pride, 1991; Fulton, Fischer, Drake, & Bromberg, 1984; Lorino et al., 1998). However, the technique has not been applied to animal nasal studies. In the current study, we evaluated changes in nasal patency in ovalbumin-sensitized guinea pigs using a modified pressure-rhinometry technique first described by Salem and Clemente (1972). In their studies, guinea pigs were anesthetized with pentobarbital. Two polyethylene cannulas were inserted into the trachea. One cannula was directed towards the lung and was used to ventilate the animal. The second cannula was directed retrograde towards the nasal cavities. The esophagus was ligated and the mouth was sealed. Air was passed through the second cannula, across the nasal cavity and out the nares at a flow rate of 1000 ml/min. The nasal pressure was derived from forced airflow across the cavities. In addition to studying nasal patency using the modified rhinometric method, we also evaluated the in-phase component of nasal impedance (i.e., nasal resistance) using forced frequency oscillations (Dubois et al., 1956; Preuss, Hall, & Sly, 1999). Changes in the out-of-phase component of nasal impedance (i.e., reactance) were minimal and were not studied. In our studies, the H1 antagonist, chlorpheniramine, and the a-agonist decongestant, phenylpropanolamine, were used as pharmacological tools to assess changes in nasal patency after nasal exposure to ovalbumin. We found the forced oscillation method useful in studying nasal resistance in guinea pigs. Moreover, we also observed that chlorpheniramine, but not phenylpropanolamine, blocked the nasal obstruction elicited by ovalbumin in allergic guinea pigs.
2. Materials and methods 2.1. Drugs Chicken egg albumin (ovalbumin), chlorpheniramine maleate, pyrilamine maleate, and phenylpropanolamine hydrochloride were purchased from Sigma (St. Louis, MO). Aluminum hydroxide was purchased from Reheis (Berkeley Heights, NJ) and pentobarbital sodium was purchased from Abbott Laboratories (Chicago, IL). Drug doses refer to their respective free bases. All drugs were dissolved in physiological saline (0.9%). 2.2. Animal care and use These studies were performed in accordance with the NIH Guide to the Care and Use of Laboratory Animals and the Animal Welfare Act in an AAALAC-accredited facility. 2.3. Sensitization of guinea pigs Male Hartley guinea pigs (300 – 350 g, Charles River, Bloomington, MA) were actively sensitized to ovalbumin over a 27-day regimen. On Day 1, animals were administered ovalbumin (100 Ag ip) and aluminum hydroxide (200 mg ip) suspended in 0.5 ml of water. On Day 7, animals were administered an additional dose of ovalbumin (100 Ag ip). The animals were used 27 days after the initial ovalbumin dose when they weighed between 450 and 500 g. 2.4. General For all studies, male ovalbumin-sensitized Hartley guinea pigs (450 – 500 g, Charles River) were used. The guinea pigs were anesthetized with pentobarbital sodium (50 mg/ kg ip). A 2.5-cm incision was made in the ventral neck region. The trachea and the right jugular vein were isolated. The trachea was cannulated with polyethylene tubing (PE240) and the animals were mechanically ventilated (volume = 0.004 l, rate = 55 breaths/min) with room air using a Harvard rodent ventilator (South Natick, MA). The jugular vein was cannulated for the administration of intravenous drugs. All experiments were conducted with the guinea pigs in the supine position. 2.5. Evaluation of nasal resistance in guinea pigs Changes in nasal resistance in ovalbumin-sensitized guinea pigs were determined according to modified methods of Salem and Clemente (1972). A 1.0-cm segment of the esophagus (approximately 2.0 cm from the oral cavity) was isolated. A small surgical incision was made into the esophagus and PE-240 was inserted and advanced to the nasopharynx. The sectioned distal end of the esophagus was ligated with 3-0 suture silk. The right naris and the oral cavity were sealed with 0.5 –1.0 ml of reprosil (DENTSPLY
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International, Milford, DE) to prevent air leakage. Humidified air (0.6 l/min at room temperature) was passed through the esophageal cannula and out through the left nasal cavity. Pressure changes across the cavity were measured through a port off of the esophageal cannula using a physiological pressure transducer (P23XL, Viggo-Spectramed, Oxnard, CA) and recorded continuously on a Grass-chart recorder. Nasal pressure values were converted to nasal resistance using the formula: resistance = D pressure/flow. Ovalbumin (3%) was aerosolized using an Ultra-NeB 99 Devilbiss nebulizer (Somerset, PA) and was passed through the esophageal cannula across the nasal cavity. The exposure duration for the challenge was 10 min. Nasal resistances were determined immediately after the ovalbumin challenge at 5-min intervals after ovalbumin challenge. We evaluated the effect of histamine H1 antagonists chlorpheniramine (1 mg/kg iv) and pyrilamine (1 mg/kg iv) and the a-adrenergic agonist, phenylpropanolamine (3 mg/kg iv), on the increase in nasal resistance produced by nasal ovalbumin exposure. Test drugs were given 10 min before ovalbumin challenge. The doses of test drugs were chosen based on literature data demonstrating pharmacological activity (McLeod, Mingo, Herczku, DeGennaro-Culver, et al., 1999; McLeod, Mingo, Herczku, Corboz, et al., 1999).
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ume = 0.002 l, 40 – 50 strokes/min). The nasal cavity was exposed to aerosolized ovalbumin through the nasal catheter for 10 min. We evaluated the nasal effect of graded concentrations of ovalbumin (0.3 – 3.0%, for 10 min) on nasal resistance. In separate animals, the decongestant activity of chlorpheniramine (1 mg/kg iv) and phenylpropanolamine (3 mg/kg iv) was studied. Chlorpheniramine and phenylpropanolamine were given 10 min before ovalbumin challenge. 2.7. Statistics The data are expressed as percent increase in nasal resistance over baseline values. The spectral frequency analysis data are presented as resistance values (cm H2O l/ min) plotted against frequency (1– 18 Hz). Calculation of the area under the frequency curve (AUC) was determined for each treatment group and is displayed in Fig. 4. The figures represent the mean F S.E.M. of 4– 10 animals per group. Data were evaluated using a nonparametric Kruskal –
2.6. Evaluation of nasal resistance using forced oscillations The right nasal cavity was cannulated with a 1.5-cmlong PE-240 tube. The tip of the cannula was advanced 1.5 –2.0 mm into the nares. The nasal cavity was sealed around the catheter with reprosil to form an airtight fit. The oral cavity was also sealed with reprosil. After placement of the trachea cannula to support mechanical ventilation, the anterior open end of the trachea was ligated, closed with surgical suture to prevent pressure drops from the nasal cavity. Likewise, the esophagus was also ligated to prevent airflow into the digestive tract. The open end of the nasal catheter was connected to a small animal ventilator and respiratory mechanics analyzer (FlexiVent, SCIREQ, Montreal, Canada). We modified the forced oscillation technique previously described by Bates, Schuessler, Dolman, and Eidelman (1997) to evaluate nasal patency. Oscillatory peak-to-peak pressure swings of 0.58 ml were generated by a piston pump. Air passed from the FlexiVent ventilator through the right nasal cavity and out the left naris. Changes in nasal resistance were evaluated at 2 Hz using software obtained from SCIREQ. Additionally, nasal resistance was measured simultaneously at 10 mutually prime oscillation frequencies ranging from 1.25 to 18.25 Hz using programmed pseudorandom noise termed primewaves. Primewaves were measured over 16 s. Resistance measurements were obtained using solid-state pressure transducers supplied by SCIREQ with a frequency response of 100 Hz. In between resistance measurements, the nasal passages were ventilated with air by the FlexiVent ventilator (vol-
Fig. 1. Effect of intravenous chlorpheniramine, pyrilamine, and phenylpropanolamine on the increase in nasal resistance produced by aerosolized ovalbumin challenge in sensitized animals. (A) Time-course effect of saline (10 min, n = 8) and ovalbumin (3%; 10 min, n = 8) evaluated by a modified rhinometric technique. (B) Effects of chlorpheniramine (CTM; 1 mg/kg iv, n = 6), pyrilamine (PYRM; 1 mg/kg iv, n = 4), and phenylpropanolamine (PPA; 3 mg/kg iv, n = 6) on the increase in nasal resistance at 30 min after intranasal ovalbumin (3%) challenge. Each point or bar represents the mean F S.E.M. * P < .05 compared to control animals; * * P < .05 compared to vehicle-treated animals.
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Wallis analysis in conjunction with a Mann – Whitney U test. Statistical significance was set at P < .05.
3. Results 3.1. Nasal resistance in guinea pigs The mean baseline nasal resistance evaluated by the modified rhinometric method for sensitized control animals was 7.9 F 0.9 cm H2Ol/min. This value was not different from the baseline resistance values of animals from the treatment groups, which ranged from 7.6 F 0.7 to 10.7 F 1.5 cm H2Ol/min. The effects of physiological buffered saline and ovalbumin 3% aerosolized through the left nasal passageway of control animals are shown in Fig. 1A. Saline produced a transient increase in nasal resistance immediately after exposure, however, this effect dissipated after 3 min. Nasal exposure to ovalbumin increased nasal resistance throughout the 30-min observation period (Fig. 1A). The maximum nasal obstruction effect of ovalbumin was seen between 20 and 30 min. Fig. 1B illustrates the activity of
Fig. 3. Forced oscillation frequency (1 – 18 Hz) analysis of the effect of intravenous chlorpheniramine on the increase in nasal resistance produced by aerosolized ovalbumin challenge in sensitized animals. Shown are the resistances of control (n = 10), ovalbumin challenged (n = 10), and ovalbumin-challenged animals treated with chlorpheniramine (1 mg/kg iv, n = 8) at baseline (A) and 30 min after ovalbumin nasal challenge (B). Each point represents the mean F S.E.M. * P < .05 compared to control animals; * * P < .05 compared to vehicle-treated animals.
chlorpheniramine (1 mg/kg iv), pyrilamine (1 mg/kg iv), and phenylpropanolamine (3 mg/kg iv) on the increase in nasal resistance elicited by ovalbumin. Both chlorpheniramine and pyrilamine significantly blocked the nasal effect of ovalbumin. Increases in nasal resistances due to ovalbumin exposure in the chlorpheniramine- and pyrilaminetreated groups were not different from control-treated animals. Phenylpropanolamine (3 mg/kg iv), however, did not attenuate the nasal-obstructive activity of ovalbumin. The percent increase in nasal resistance over baseline values at 30 min were 50 F 17%, 39 F 11%, 95 F 22%, and 99 F 14% for the chlorpheniramine, pyrilamine, phenylpropanolamine, and vehicle treatment groups, respectively. 3.2. Evaluation of nasal resistance using forced oscillations Fig. 2. Low-frequency 2-Hz forced oscillation analysis of the effect of intravenous chlorpheniramine and phenylpropanolamine on the increase in nasal resistance produced by aerosolized ovalbumin challenge in sensitized animals. (A) Time-course effect of saline (10 min, n = 10) and ovalbumin (3%; 10 min, n = 10) on nasal resistance. (B) Effects of chlorpheniramine (CTM; 1 mg/kg iv, n = 8) and phenylpropanolamine (PPA; 3 mg/kg iv, n = 8) at 30 min on the increase in nasal resistance produced by nasal ovalbumin (3%). Each point or bar represents the mean F S.E.M. * P < .05 compared to control animals; * * P < .05 compared to vehicle-treated animals.
Fig. 2A illustrates the time-course changes in nasal resistance produced by saline and ovalbumin (3%) at a singleoscillation frequency of 2 Hz. The mean baseline resistance value for the control group was 1.0 F 0.03 cm H2Ol/min. Baseline resistance values for the treatment groups were not statistically different from control animals. Aerosolized ovalbumin increased nasal resistance compared to the control
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group, and the maximum increase in nasal resistance of 91 F 14% elicited by ovalbumin was observed 30 min after exposure. Fig. 2B shows that chlorpheniramine (1 mg/kg iv), but not phenylpropanolamine (3 mg/kg iv), blocked the increase in nasal resistance at 2 Hz produced by ovalbumin. Nasal resistances in the chlorpheniramine group after ovalbumin challenge was not different from control-treated animals. In addition, phenylpropanolamine, given at a higher dose of 10 mg/kg iv, did not block the nasal effect of ovalbumin (n = 7; data not shown). The complete 1- to 18Hz frequency assessment for the changes in nasal resistance due to nasal provocation to ovalbumin is displayed in Fig. 3. There was no difference in baseline nasal resistance values between treatment groups (Fig. 3A). Fig. 3B shows that chlorpheniramine (1 mg/kg iv) blocked the increase in nasal resistance elicited by ovalbumin when evaluated by spectral analysis. Calculation of AUC for frequencies ranging from 1 to 18 Hz demonstrates that ovalbumin (0.3 – 3.0%) produced a dose-dependent increase in nasal resistance (Fig. 4A). Moreover, the AUC(1 – 18 Hz) at 30 min for the ovalbumin (1% and 3%) were 1.77 F 0.18 and 3.18 F 0.72 cm H2Ol/ minHz and were significantly different from control animals,
Fig. 4. Area under complete frequency (1 – 18 Hz) curve analysis. (A) AUC for controls (n = 10) and ovalbumin-challenged (n = 10) animals. (B) AUC for controls and ovalbumin-challenged animals treated with chlorpheniramine (1 mg/kg iv, n = 8) and phenylpropanolamine (3 mg/kg iv, n = 8) at baseline and 30 min after ovalbumin nasal challenge. Each bar represents the mean F S.E.M. * P < .05 compared to control animals; * * P < .05 compared to vehicle-treated animals.
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0.78 F 0.18 cm H2Ol/minHz. The AUC(1 – 18 Hz) analysis also shows that chlorpheniramine (1 mg/kg iv), but not phenylpropanolamine (3 mg/kg iv), blocked allergic-induced nasal obstruction (Fig. 4B).
4. Discussion The forced oscillation method permits measurements of respiratory system impedance and its components as a function of frequency. The method has been used to study pulmonary mechanics in animals (Clercx, Gustin, Landser, & Van de Woestijne, 1993; Petak, Hall, & Sly, 1998; Preuss et al., 1999) and man (Delacourt et al., 2000; Farre, Peslin, Rotger, Barbera, & Navajas, 1999; Hall, Hantos, Wildhaber, Petak, & Sly, 2001). Moreover, the results generated by the forced oscillation technique are consistent with data generated by other methods used to evaluate pulmonary obstruction and bronchial reactivity (Farre et al., 1999). Only a few reports have described the use of the forced oscillation technique in human nasal studies (Aksamit et al., 1991; Fulton et al., 1984; Lorino et al., 1998). To date, the utility of the forced oscillation technique in animal nasal studies has not been discussed. In the present investigation, we studied the increases in nasal airway resistance produced by intranasally administered ovalbumin to sensitized guinea pigs using a modified rhinometric method based on that described by Salem and Clemente (1972). In addition, we evaluated nasal patency using the forced oscillation method. Interesting, baseline guinea pig nasal resistance values have not been extensively reported. Nevertheless, we found, using the modified rhinometric method, that our baseline nasal resistance values (7.9 F 0.9 cm H2Ol/min) for control animals were less than the conscious guinea pig nasal baseline resistance values (15 F 1.7 cm H2Ol/min) reported by Fujita et al. (1999). It must be pointed out that Fujita et al. used a plethysmographic technique to study nasal patency. Consequently, measurements of nasal resistance were not exclusively limited to an evaluation of upper-airway patency but also incorporated an assessment of pulmonary resistance. Curran, O’Halloran, and Bradford (1998) on the other hand, used a rhinometric-based technique to study the effects of cold air on nasal resistance in anesthetized guinea pigs. These investigators reported baseline nasal resistance values of approximately 0.21 cm H2Ol/min in this species. Therefore, it appears that baseline nasal resistances in the guinea pig may vary significantly depending on the method utilized to determine this respiratory parameter. In fact, we found that fundamental differences in the delivery of air (and airflow rates) to the nasal cavity between our modified rhinometric method and the forced oscillation method did not allow us to directly compare baseline nasal resistance values obtained from each method. For example, using the rhinometric method, a cannula was placed into the esophagus and positioned next to the nasopharynx. This positioning allowed retrograde airflow across the nasal cavity. In
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contrast, in the forced oscillation method, air was delivered orthograde through a nasal cannula and resistance was determined across one nares. Nevertheless, we found that the qualitative results from each of the methods were not different. For example, the increase in nasal resistance produced by 3% ovalbumin at 30 min posttreatment was 99 F 14% and 91 F 14% for the rhinometric and the forced oscillation methods, respectively. The forced oscillation technique permits the measurement of airway mechanics over a wide-frequency range. The data can then be fitted to theoretical models of the respiratory system to identify which elements of the airway are responsible for the changes in mechanics. Many models of the respiratory system have been proposed, but the constant phase model of Petak, Hantos, Adamicza, Asztalos, and Sly (1997) fits experimental data well over a range of physiological and pathological states with a small number of model elements. In contrast, there are no models of the nasal system to fit to forced oscillation data and we did not attempt such an analysis. However, the relatively flat resistance –frequency graph (Fig. 3) suggests that there is not much additional information to be gained by making measurements of resistance over the frequency range 1 –18 Hz as compared to a single-spot frequency. This is borne out by the similarities between Figs. 2B and 4B. We found that the histamine H1 antagonist chlorpheniramine blocked the nasal obstruction produced by ovalbumin in sensitized guinea pigs. The nasal activity of chlorpheniramine was demonstrated using both the modified rhinometric method and the forced oscillation technique. A second H1 antagonist tested, pyrilamine, was also found to block the obstruction produced by nasal exposure to ovalbumin challenge. Our results with chlorpheniramine and pyrilamine are consistent with previous studies showing that H1 antagonists block nasal obstruction in allergic guinea pigs (Narita & Asakura, 1993; Ohkawa, Ukai, Miyahara, Takeuchi, & Sakakura, 1999). In contrast to the effects of chlorpheniramine and pyrilamine, we found that phenylpropanolamine did not block nasal obstruction. Our results with phenylpropanolamine are not consistent with the findings of Imai et al. (2001), denoting that a-agonists produce nasal decongestion in the guinea pig. Nevertheless, interpretation of our finding suggests that nasal vasodilatation does not appear to significantly contribute to allergic nasal obstruction in the guinea pig. Consequently, we propose that nasal obstruction in the allergic guinea pig may be distinct from nasal congestion in man. Unquestionably, a-adrenergic agonist decongestants ameliorate nasal vascular engorgement characteristic of human nasal congestion. Furthermore, a-agonist decongestants also effectively improve nasal patency in the cat, dog, and pig (Koss, Yu, Hey, & McLeod, 2002a,b; Lacroix, 1989; McLeod, Mingo, Herczku, DeGennaro-Culver, et al., 1999; McLeod, Mingo, Herczku, Corboz, et al., 1999). Allergy-induced nasal obstruction in the guinea pig is likely due to a histamine H1 receptor-mediated increase in nasal vascular permeability
and an increase in glandular secretions (Gawin, Baraniuk, & Kaliner, 1992). A second distinction between guinea pig nasal obstruction and human nasal congestion is that in man, H1 antagonists are not very effective decongestants (Spector, 1999). The differences in the nasal pharmacological effects of the H1 antagonists and the a-decongestants in the guinea pig and man may be related to differences in the morphological organization of upper airways among the two species. In support of this hypothesis, using conventional light and electron microscopy, Pastor, Amores, Villaverde, Calvo, and Spreklsen (1990) identified two distinct nasal conchae in the guinea pig, namely, the nasal turbinate and the maxilloturbinate. In humans, typically three turbinates (inferior, middle, and superior) are found. In addition, Pastor et al. also found that the anterior sections of guinea pig turbinates, like human turbinates, are highly vascularized. However, in contrast to findings in humans, the anterior portion of the guinea pig turbinate does not contain glands. Nasal glands in the guinea pig nasal cavity are distributed in more posterior sections of the nasal turbinates. Another significant distinction between guinea pigs and humans relates to the functionality of the upper respiratory system. For example, it should be pointed out that humans and most mammals have the capacity to breathe through their nose as well as their mouth. On the other hand, guinea pigs and most rodents are nasal-obligatory breathers. The extent to which these distinct morphological and functional differences in the upper respiratory airways of guinea pigs relative to humans affect nasal physiology remains to be determined. Nevertheless, a consideration of the unique features of the guinea pig nose should be considered when this species is used to extrapolate nasal physiology and pharmacology to humans. In summary, we characterized the nasal-decongestant effects of H1 antagonists and the a-adrenergic agonist, phenylpropanolamine, using two different methods in the guinea pig. In both of these models, the H1 antagonists significantly blocked ovalbumin-induced nasal obstruction; however, the a-agonist produced little or no effect on allergic nasal obstruction. In light of the present findings, we suggest that additional studies to fully characterize the nasal effects of a-adrenergic agonists in the guinea pig are warranted. Furthermore, it is important to recognize the utility of the forced oscillation method as an effective technique in evaluating nasal patency in preclinical animal models. References Aksamit, T., Duggan, C., Watson, A., & Pride, N. D. (1991). Use of oscillation methods to measure nasal airflow resistance. European Respiratory Review, 1, 232 – 235. Albert, D. H., Malo, P. E., Tapang, P., Shaughnessy, T. K., Morgan, D. W., Wegner, C. D., Curtin, M. L., Sheppard, G. S., Xu, L., Davidsen, S. K., Summers, J. B., & Carter, G. W. (1998). The role of platelet-activating factor (PAF) and the efficacy of ABT-491, a highly potent and selective PAF antagonist, in experimental allergic rhinitis. Journal of Pharmacology and Experimental Therapeutics, 284, 83 – 88.
R.L. McLeod et al. / Journal of Pharmacological and Toxicological Methods 48 (2002) 153–159 ¨ nggard, A., & Densert, O. (1974). Adrenergic innervation of the nasal A mucosa in cat. A histological and physiological study. Acta OtoLaryngologica, 78(3 – 4), 232 – 241. Bates, J. H. T., Schuessler, T. F., Dolman, C., & Eidelman, D. H. (1997). Temporal dynamics of acute isovolume bronchoconstriction in the rat. Journal of Applied Physiology, 82(1), 55 – 62. Benazzo, M., Casasco, A., Lovotti, P., Icaro Cornaglia, A., Casasco, M., & Polak, J. M. (1994). Endothelin-induced vasoconstriction in rabbit nasal mucosa. Acta Oto-Laryngologica, 114(5), 544 – 546. Bende, M., Hansell, P., Intaglietta, M., & Arfors, K. E. (1992). Effect of oxymetazoline nose drops on vascular permeability of the nasal mucosa in the rabbit after provocation with leukotriene B4. ORL. Journal of Oto-Rhino-Laryngology and Its Related Specialties, 54(5), 270 – 274. Clercx, C., Gustin, P., Landser, F. J., & Van de Woestijne, K. P. (1993). Measurement of total respiratory impedance in dogs by the forced oscillation technique. Veterinary Research Communications, 17(3), 227 – 239. Cole, P., Roithmann, R., Roth, Y., & Chapnik, J. S. (1997). Measurement of airway patency. Annals of Otology, Rhinology & Laryngology. Supplement, 171, 1 – 23. Curran, A. K., O’Halloran, K. D., & Bradford, A. (1998). Upper airway cooling reduces upper airway resistance in anesthetized young guinea pigs. European Respiratory Journal, 11, 1257 – 1262. Delacourt, C., Lorino, H., Herve-Guillot, M., Reinert, P., Harf, A., & Housset, B. (2000). Use of the forced oscillation technique to assess airway obstruction and reversibility in children. American Journal of Respiratory and Critical Care Medicine, 161(3 Pt 1), 730 – 736. Dubois, A. B., Brody, A. W., Lewis, D. H., & Burgess, B. F. (1956). Oscillation mechanics of lungs and chest in man. Journal of Applied Physiology, 8, 587 – 594. Dylewska, K., Sahin, G., & Widdicombe, J. G. (1993). Asymmetric reflex responses of the nasal and tracheal vasculatures of the dog. Journal of Applied Physiology, 75(5), 2157 – 2161. Eccles, R. (1978). The domestic pig as an experimental model for studies on the nasal cycle. Acta Oto-Laryngologica, 85(5 – 6), 431 – 436. Eccles, R., & Wilson, H. (1974). The autonomic innervation of the nasal blood vessels of the cat. Journal of Physiology, 238(3), 549 – 560. Erickson, C. H., McLeod, R. L., Mingo, G. G., Egan, R. W., Pedersen, O. F., & Hey, J. A. (2001). Comparative oral and topical decongestant effects of phenylpropanolamine and D-pseudoephedrine. American Journal of Rhinology, 15(2), 83 – 90. Farre, R., Peslin, R., Rotger, M., Barbera, J. A., & Navajas, D. (1999). Forced oscillation total respiratory resistance and spontaneous breathing lung resistance in COPD patients. European Respiratory Journal, 14(1), 172 – 178. Fujita, M., Yonetomi, Y., Shimouchi, K., Aze, Y., Kawabata, K., & Ohno, H. (1999). Involvement of cysteinyl leukotrienes in biphasic increase of nasal airway resistance of antigen-induced rhinitis in guinea pigs. European Respiratory Journal, 369, 349 – 356. Fulton, J. M., Fischer, N. D., Drake, A. F., & Bromberg, P. A. (1984). Frequency dependence of effective nasal resistance. Annals of Otology, Rhinology & Laryngology, 93, 140 – 145. Gawin, A. Z., Baraniuk, J. N., & Kaliner, M. (1992). Effects of histamine on guinea pig nasal mucosal secretion. American Journal of Physiology, 262(5 Pt 1), L590 – L599. Hall, G. L., Hantos, Z., Wildhaber, J. H., Petak, F., & Sly, P. D. (2001). Methacholine responsiveness in infants assessed with low frequency forced oscillation and forced expiration techniques. Thorax, 56(1), 42 – 47. Imai, I., Nabe, T., Mizutani, N., Sakurai, H., Takenaka, H., & Kohno, S. (2001). Involvement of nitric oxide in pollen-induced biphasic nasal blockage in sensitized guinea pigs. European Journal of Pharmacology, 423, 63 – 70. Koss, M. C., Yu, Y., Hey, J. A., & McLeod, R. L. (2002a). Acoustic rhin-
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ometry in the dog: A novel large animal model for studies of nasal congestion. American Journal of Rhinology, 16, 49 – 55. Koss, M. C., Yu, Y., Hey, J. A., & McLeod, R. L. (2002b). Measurement of nasal patency in anesthetized and conscious dogs. Journal of Applied Physiology, 92, 617 – 621. Lacroix, J. S. (1989). Adrenergic and non-adrenergic mechanisms in sympathetic vascular control of the nasal mucosa. Acta Physiologica Scandinavica. Supplementum, 581, 1 – 63. Lacroix, J. S., Ulman, L. G., & Potter, E. K. (1994). The role of ATP in nonadrenergic sympathetic vascular control of the nasal mucosa in anaesthetized cats and dogs. Journal of Physiology, 476(3), 429 – 435. Lau, W. A. K., King, R. G., & Boura, A. L. A. (1990). Methoxyphenamine inhibits basal and histamine-induced nasal congestion in anaesthetized rats. British Journal of Pharmacology, 101, 394 – 398. Lorino, A. M., Lofaso, F., Duizabo, D., Zerah, F., Goldenberg, F., d’Ortho, M. P., Harf, A., & Lorino, H. (1998). Respiratory resistive impedance as an index of airway obstruction during nasal continuous positive airway pressure titration. American Journal of Respiratory and Critical Care Medicine, 158(5 Pt 1), 1465 – 1470. Lung, M. A., Phipps, R. J., Wang, J. C. C., & Widdicombe, J. G. (1984). Control of nasal vasculature and airflow resistance in the dog. Journal of Physiology, 349, 535 – 551. Malm, L. (1973). Stimulation of sympathetic nerve fibers to the nose in cats. Acta Oto-Laryngologica, 75, 519 – 526. McLeod, R. L., Mingo, G. G., Herczku, C., Corboz, M. R., Ramos, S. I., DeGennaro-Culver, F., Pedersen, O., & Hey, J. A. (1999). Changes in nasal resistance and nasal geometry in a feline model of nasal congestion. American Journal of Rhinology, 13(5), 375 – 383. McLeod, R. L., Mingo, G. G., Herczku, C., DeGennaro-Culver, F., Kreutner, W., Egan, R. W., & Hey, J. A. (1999). Combined histamine H1 and H3 receptor blockade produces nasal decongestion in an experimental model of nasal congestion. American Journal of Rhinology, 13(5), 391 – 399. Mizuno, H., Kawamura, Y., Iwase, N., & Ohno, H. (1991). Effects of flutropium on experimental models of drug- and allergy-induced rhinitis in guinea pigs. Japanese Journal of Pharmacology, 55(3), 321 – 328. Narita, S., & Asakura, K. (1993). The effects of anti-PAF and other agents on the nasal symptoms in sensitized guinea pigs. Auris, Nasus, Larynx, 20(3), 175 – 183. Ohkawa, C., Ukai, K., Miyahara, Y., Takeuchi, K., & Sakakura, Y. (1999). Histamine H1 receptor and reactivity of the nasal mucosa in sensitized guinea pigs. Auris, Nasus, Larynx, 26(3), 293 – 298. Pastor, L. M., Amores, A. E., Villaverde, R., Calvo, A., & Spreklsen, C. (1990). Morphological study of the nasal conchae of the guinea pig. Acta Anatomica, 139, 254 – 264. Petak, F., Hantos, Z., Adamicza, A., Asztalos, T., & Sly, P. D. (1997). Methacholine-induced bronchoconstriction in rats: Effects of intravenous vs. aerosol delivery. Journal of Applied Physiology, 82(5), 1479 – 1487. Petak, F., Hall, G. L., & Sly, P. D. (1998). Repeated measurements of airway and parenchymal mechanics in rats by using low-frequency oscillations. Journal of Applied Physiology, 84(5), 1680 – 1686. Preuss, J. M. H., Hall, G. L., & Sly, P. D. (1999). Repeat measurement of respiratory mechanics using the forced oscillation technique in nonparalyzed rats. Pulmonary Pharmacology & Therapeutics, 12, 173 – 181. Salem, H., & Clemente, E. (1972). A new experimental method for evaluating drugs in the nasal cavity. Archives of Otolaryngology, 96(6), 524 – 529. Spector, S. (1999). Ideal pharmacotherapy for allergic rhinitis. Journal of Allergy and Clinical Immunology, 103(3 Pt 2), S386 – S387. Watson, J. W., Jackson, A. C., & Drazen, J. M. (1986). Effect of lung volume on pulmonary mechanics in guinea pigs. Journal of Applied Physiology, 61(1), 304 – 311.
Original article
Characterization of nasal obstruction in the allergic guinea pig using the forced oscillation method Robbie L. McLeoda,*, Simon S. Youngb, Christine H. Ericksona, Leonard E. Parraa, John A. Heya a
Department of Allergy, Schering-Plough Research Institute, 2015 Galloping Hill Road Kenilworth, NJ 07033-0539, USA b Discovery Bioscience, AstraZeneca, Bakewell Road, Loughborough, Leicestershire LE11 5RH, UK Received 13 March 2002; accepted 20 June 2003
Abstract Introduction: This is the first report to evaluate changes in nasal resistance in a preclinical animal model using the forced oscillation method. Methods: The method involves characterizing pressure – flow relationships of the respiratory system due to external oscillatory forces. Results: First, we evaluated changes in nasal resistance using an established small-animal rhinometric technique. In these studies, aerosolized ovalbumin (3%) administered to the nasal cavity of ovalbumin-sensitized guinea pigs increased nasal resistance at 30 min by 99 F 14%. The histamine H1 antagonists, chlorpheniramine (1 mg/kg iv) and pyrilamine (1 mg/kg iv), blocked the increase in nasal resistance due to ovalbumin provocation (50 F 17% and 39 F 11% over baseline, respectively). The a-adrenergic agonist phenylpropanolamine (3 mg/ kg iv) had no effect on the nasal actions of ovalbumin. In separate studies, nasal resistance was measured at 2 Hz by forced oscillation and ovalbumin (3%) increased nasal resistance by 91 F 14%. Chlorpheniramine (1 mg/kg iv) significantly attenuated the increase in nasal resistance due to ovalbumin. Finally, changes in nasal resistance for each treatment group were evaluated at frequencies of 1 – 18 Hz. Area under the curve analysis demonstrated that chlorpheniramine blocked the nasal obstructive effect of ovalbumin. In contrast, a pharmacologically active dose of phenylpropanolamine (3 mg/kg iv) did not produce decongestant activity. Discussion: The current data are inconsistent with the well-established clinical efficacy of a-adrenergic agonists as nasal decongestants. Consequently, we suggest that allergic nasal obstruction in the guinea pig may not be the best preclinical approach to assess the nasal decongestant activity of vasoconstrictor aadrenergic agonists. Additionally, our studies demonstrate the utility of the forced oscillation technique in assessing changes in nasal resistance in small laboratory animals. D 2003 Elsevier Inc. All rights reserved. Keywords: Forced oscillation; Guinea pig; Nasal congestion; Nasal obstruction; Nasal resistance; Phenylpropanolamine
1. Introduction The ability to reliably evaluate changes in nasal cavity patency in animals is of paramount importance in the preclinical evaluation of novel treatments for upper respiratory diseases, such as allergic rhinitis. The use of small animals, such as rat (Albert et al., 1998; Lau, King, & Boura, 1990), guinea pig (Narita & Asakura, 1993), rabbit (Benazzo et al., 1994; Bende, Hansell, Intaglietta, & ¨ nggard & Densert, 1974; Eccles & Arfors, 1992), cat (A Wilson, 1974; Malm, 1973; McLeod, Mingo, Herczku, DeGennaro-Culver, et al., 1999; McLeod, Mingo, Herczku,
* Corresponding author. Tel.: +1-908 740-3286; fax: +1-908-7407175. E-mail address: [email protected] (R.L. McLeod). 1056-8719/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1056-8719(03)00044-3
Corboz, et al., 1999), and large animals, such as dog (Dylewska, Sahin, & Widdicombe, 1993; Lacroix, Ulman, & Potter, 1994; Lung, Phipps, Wang, & Widdicombe, 1984) and pig (Eccles, 1978; Lacroix, 1989), have been used to generate important information on nasal physiology and pathophysiology. Moreover, many of these models have been beneficial in assessing the pharmacology of drugs used in the treatment of nasal diseases (Mizuno, Kawamura, Iwase, & Ohno, 1991). Current techniques used to study changes in nasal patency fall into one of two categories (Cole, Roithmann, Roth, & Chapnik, 1997). Dynamic techniques derive nasal resistance from measurements of nasal airflow and differential pressures between proximal and distal points of the nasal cavity. Static methods measure nasal cavity geometry independent of airflow. Both dynamic rhinomanometric methods that evaluate nasal airway resistance and static procedures, such as
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acoustic rhinometry are, approaches that have been employed to study nasal patency in animals (Erickson et al., 2001; McLeod, Mingo, Herczku, DeGennaro-Culver, et al., 1999; McLeod, Mingo, Herczku, Corboz, et al., 1999; Koss, Yu, Hey, & McLeod, 2002a,b; Salem & Clemente, 1972). Additionally, there are a few methods especially suited to study changes in nasal resistances in small laboratory animals, such as guinea pigs. However, none of these methods are ideal. For example, plethysmographic methods measure total respiratory resistance (pulmonary and nasal). Consequently, using this method, it is difficult to characterize changes in resistances due solely to changes in nasal patency. Therefore, characterizing new and potentially improved methods for evaluating nasal resistance, such as the forced oscillation technique, may be important for preclinical research and the development of novel therapies. The forced oscillation method was first introduced by Dubois, Brody, Lewis, and Burgess (1956) to study pulmonary mechanics. The technique involves characterizing the pressure – flow relationship of the respiratory system due to external oscillatory forces. Estimates of pulmonary resistance and reactance are determined as a function of frequency (Watson, Jackson, & Drazen, 1986). A few clinical investigators have applied the forced oscillation method to assess nasal resistance in humans (Aksamit, Duggan, Watson, & Pride, 1991; Fulton, Fischer, Drake, & Bromberg, 1984; Lorino et al., 1998). However, the technique has not been applied to animal nasal studies. In the current study, we evaluated changes in nasal patency in ovalbumin-sensitized guinea pigs using a modified pressure-rhinometry technique first described by Salem and Clemente (1972). In their studies, guinea pigs were anesthetized with pentobarbital. Two polyethylene cannulas were inserted into the trachea. One cannula was directed towards the lung and was used to ventilate the animal. The second cannula was directed retrograde towards the nasal cavities. The esophagus was ligated and the mouth was sealed. Air was passed through the second cannula, across the nasal cavity and out the nares at a flow rate of 1000 ml/min. The nasal pressure was derived from forced airflow across the cavities. In addition to studying nasal patency using the modified rhinometric method, we also evaluated the in-phase component of nasal impedance (i.e., nasal resistance) using forced frequency oscillations (Dubois et al., 1956; Preuss, Hall, & Sly, 1999). Changes in the out-of-phase component of nasal impedance (i.e., reactance) were minimal and were not studied. In our studies, the H1 antagonist, chlorpheniramine, and the a-agonist decongestant, phenylpropanolamine, were used as pharmacological tools to assess changes in nasal patency after nasal exposure to ovalbumin. We found the forced oscillation method useful in studying nasal resistance in guinea pigs. Moreover, we also observed that chlorpheniramine, but not phenylpropanolamine, blocked the nasal obstruction elicited by ovalbumin in allergic guinea pigs.
2. Materials and methods 2.1. Drugs Chicken egg albumin (ovalbumin), chlorpheniramine maleate, pyrilamine maleate, and phenylpropanolamine hydrochloride were purchased from Sigma (St. Louis, MO). Aluminum hydroxide was purchased from Reheis (Berkeley Heights, NJ) and pentobarbital sodium was purchased from Abbott Laboratories (Chicago, IL). Drug doses refer to their respective free bases. All drugs were dissolved in physiological saline (0.9%). 2.2. Animal care and use These studies were performed in accordance with the NIH Guide to the Care and Use of Laboratory Animals and the Animal Welfare Act in an AAALAC-accredited facility. 2.3. Sensitization of guinea pigs Male Hartley guinea pigs (300 – 350 g, Charles River, Bloomington, MA) were actively sensitized to ovalbumin over a 27-day regimen. On Day 1, animals were administered ovalbumin (100 Ag ip) and aluminum hydroxide (200 mg ip) suspended in 0.5 ml of water. On Day 7, animals were administered an additional dose of ovalbumin (100 Ag ip). The animals were used 27 days after the initial ovalbumin dose when they weighed between 450 and 500 g. 2.4. General For all studies, male ovalbumin-sensitized Hartley guinea pigs (450 – 500 g, Charles River) were used. The guinea pigs were anesthetized with pentobarbital sodium (50 mg/ kg ip). A 2.5-cm incision was made in the ventral neck region. The trachea and the right jugular vein were isolated. The trachea was cannulated with polyethylene tubing (PE240) and the animals were mechanically ventilated (volume = 0.004 l, rate = 55 breaths/min) with room air using a Harvard rodent ventilator (South Natick, MA). The jugular vein was cannulated for the administration of intravenous drugs. All experiments were conducted with the guinea pigs in the supine position. 2.5. Evaluation of nasal resistance in guinea pigs Changes in nasal resistance in ovalbumin-sensitized guinea pigs were determined according to modified methods of Salem and Clemente (1972). A 1.0-cm segment of the esophagus (approximately 2.0 cm from the oral cavity) was isolated. A small surgical incision was made into the esophagus and PE-240 was inserted and advanced to the nasopharynx. The sectioned distal end of the esophagus was ligated with 3-0 suture silk. The right naris and the oral cavity were sealed with 0.5 –1.0 ml of reprosil (DENTSPLY
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International, Milford, DE) to prevent air leakage. Humidified air (0.6 l/min at room temperature) was passed through the esophageal cannula and out through the left nasal cavity. Pressure changes across the cavity were measured through a port off of the esophageal cannula using a physiological pressure transducer (P23XL, Viggo-Spectramed, Oxnard, CA) and recorded continuously on a Grass-chart recorder. Nasal pressure values were converted to nasal resistance using the formula: resistance = D pressure/flow. Ovalbumin (3%) was aerosolized using an Ultra-NeB 99 Devilbiss nebulizer (Somerset, PA) and was passed through the esophageal cannula across the nasal cavity. The exposure duration for the challenge was 10 min. Nasal resistances were determined immediately after the ovalbumin challenge at 5-min intervals after ovalbumin challenge. We evaluated the effect of histamine H1 antagonists chlorpheniramine (1 mg/kg iv) and pyrilamine (1 mg/kg iv) and the a-adrenergic agonist, phenylpropanolamine (3 mg/kg iv), on the increase in nasal resistance produced by nasal ovalbumin exposure. Test drugs were given 10 min before ovalbumin challenge. The doses of test drugs were chosen based on literature data demonstrating pharmacological activity (McLeod, Mingo, Herczku, DeGennaro-Culver, et al., 1999; McLeod, Mingo, Herczku, Corboz, et al., 1999).
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ume = 0.002 l, 40 – 50 strokes/min). The nasal cavity was exposed to aerosolized ovalbumin through the nasal catheter for 10 min. We evaluated the nasal effect of graded concentrations of ovalbumin (0.3 – 3.0%, for 10 min) on nasal resistance. In separate animals, the decongestant activity of chlorpheniramine (1 mg/kg iv) and phenylpropanolamine (3 mg/kg iv) was studied. Chlorpheniramine and phenylpropanolamine were given 10 min before ovalbumin challenge. 2.7. Statistics The data are expressed as percent increase in nasal resistance over baseline values. The spectral frequency analysis data are presented as resistance values (cm H2O l/ min) plotted against frequency (1– 18 Hz). Calculation of the area under the frequency curve (AUC) was determined for each treatment group and is displayed in Fig. 4. The figures represent the mean F S.E.M. of 4– 10 animals per group. Data were evaluated using a nonparametric Kruskal –
2.6. Evaluation of nasal resistance using forced oscillations The right nasal cavity was cannulated with a 1.5-cmlong PE-240 tube. The tip of the cannula was advanced 1.5 –2.0 mm into the nares. The nasal cavity was sealed around the catheter with reprosil to form an airtight fit. The oral cavity was also sealed with reprosil. After placement of the trachea cannula to support mechanical ventilation, the anterior open end of the trachea was ligated, closed with surgical suture to prevent pressure drops from the nasal cavity. Likewise, the esophagus was also ligated to prevent airflow into the digestive tract. The open end of the nasal catheter was connected to a small animal ventilator and respiratory mechanics analyzer (FlexiVent, SCIREQ, Montreal, Canada). We modified the forced oscillation technique previously described by Bates, Schuessler, Dolman, and Eidelman (1997) to evaluate nasal patency. Oscillatory peak-to-peak pressure swings of 0.58 ml were generated by a piston pump. Air passed from the FlexiVent ventilator through the right nasal cavity and out the left naris. Changes in nasal resistance were evaluated at 2 Hz using software obtained from SCIREQ. Additionally, nasal resistance was measured simultaneously at 10 mutually prime oscillation frequencies ranging from 1.25 to 18.25 Hz using programmed pseudorandom noise termed primewaves. Primewaves were measured over 16 s. Resistance measurements were obtained using solid-state pressure transducers supplied by SCIREQ with a frequency response of 100 Hz. In between resistance measurements, the nasal passages were ventilated with air by the FlexiVent ventilator (vol-
Fig. 1. Effect of intravenous chlorpheniramine, pyrilamine, and phenylpropanolamine on the increase in nasal resistance produced by aerosolized ovalbumin challenge in sensitized animals. (A) Time-course effect of saline (10 min, n = 8) and ovalbumin (3%; 10 min, n = 8) evaluated by a modified rhinometric technique. (B) Effects of chlorpheniramine (CTM; 1 mg/kg iv, n = 6), pyrilamine (PYRM; 1 mg/kg iv, n = 4), and phenylpropanolamine (PPA; 3 mg/kg iv, n = 6) on the increase in nasal resistance at 30 min after intranasal ovalbumin (3%) challenge. Each point or bar represents the mean F S.E.M. * P < .05 compared to control animals; * * P < .05 compared to vehicle-treated animals.
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Wallis analysis in conjunction with a Mann – Whitney U test. Statistical significance was set at P < .05.
3. Results 3.1. Nasal resistance in guinea pigs The mean baseline nasal resistance evaluated by the modified rhinometric method for sensitized control animals was 7.9 F 0.9 cm H2Ol/min. This value was not different from the baseline resistance values of animals from the treatment groups, which ranged from 7.6 F 0.7 to 10.7 F 1.5 cm H2Ol/min. The effects of physiological buffered saline and ovalbumin 3% aerosolized through the left nasal passageway of control animals are shown in Fig. 1A. Saline produced a transient increase in nasal resistance immediately after exposure, however, this effect dissipated after 3 min. Nasal exposure to ovalbumin increased nasal resistance throughout the 30-min observation period (Fig. 1A). The maximum nasal obstruction effect of ovalbumin was seen between 20 and 30 min. Fig. 1B illustrates the activity of
Fig. 3. Forced oscillation frequency (1 – 18 Hz) analysis of the effect of intravenous chlorpheniramine on the increase in nasal resistance produced by aerosolized ovalbumin challenge in sensitized animals. Shown are the resistances of control (n = 10), ovalbumin challenged (n = 10), and ovalbumin-challenged animals treated with chlorpheniramine (1 mg/kg iv, n = 8) at baseline (A) and 30 min after ovalbumin nasal challenge (B). Each point represents the mean F S.E.M. * P < .05 compared to control animals; * * P < .05 compared to vehicle-treated animals.
chlorpheniramine (1 mg/kg iv), pyrilamine (1 mg/kg iv), and phenylpropanolamine (3 mg/kg iv) on the increase in nasal resistance elicited by ovalbumin. Both chlorpheniramine and pyrilamine significantly blocked the nasal effect of ovalbumin. Increases in nasal resistances due to ovalbumin exposure in the chlorpheniramine- and pyrilaminetreated groups were not different from control-treated animals. Phenylpropanolamine (3 mg/kg iv), however, did not attenuate the nasal-obstructive activity of ovalbumin. The percent increase in nasal resistance over baseline values at 30 min were 50 F 17%, 39 F 11%, 95 F 22%, and 99 F 14% for the chlorpheniramine, pyrilamine, phenylpropanolamine, and vehicle treatment groups, respectively. 3.2. Evaluation of nasal resistance using forced oscillations Fig. 2. Low-frequency 2-Hz forced oscillation analysis of the effect of intravenous chlorpheniramine and phenylpropanolamine on the increase in nasal resistance produced by aerosolized ovalbumin challenge in sensitized animals. (A) Time-course effect of saline (10 min, n = 10) and ovalbumin (3%; 10 min, n = 10) on nasal resistance. (B) Effects of chlorpheniramine (CTM; 1 mg/kg iv, n = 8) and phenylpropanolamine (PPA; 3 mg/kg iv, n = 8) at 30 min on the increase in nasal resistance produced by nasal ovalbumin (3%). Each point or bar represents the mean F S.E.M. * P < .05 compared to control animals; * * P < .05 compared to vehicle-treated animals.
Fig. 2A illustrates the time-course changes in nasal resistance produced by saline and ovalbumin (3%) at a singleoscillation frequency of 2 Hz. The mean baseline resistance value for the control group was 1.0 F 0.03 cm H2Ol/min. Baseline resistance values for the treatment groups were not statistically different from control animals. Aerosolized ovalbumin increased nasal resistance compared to the control
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group, and the maximum increase in nasal resistance of 91 F 14% elicited by ovalbumin was observed 30 min after exposure. Fig. 2B shows that chlorpheniramine (1 mg/kg iv), but not phenylpropanolamine (3 mg/kg iv), blocked the increase in nasal resistance at 2 Hz produced by ovalbumin. Nasal resistances in the chlorpheniramine group after ovalbumin challenge was not different from control-treated animals. In addition, phenylpropanolamine, given at a higher dose of 10 mg/kg iv, did not block the nasal effect of ovalbumin (n = 7; data not shown). The complete 1- to 18Hz frequency assessment for the changes in nasal resistance due to nasal provocation to ovalbumin is displayed in Fig. 3. There was no difference in baseline nasal resistance values between treatment groups (Fig. 3A). Fig. 3B shows that chlorpheniramine (1 mg/kg iv) blocked the increase in nasal resistance elicited by ovalbumin when evaluated by spectral analysis. Calculation of AUC for frequencies ranging from 1 to 18 Hz demonstrates that ovalbumin (0.3 – 3.0%) produced a dose-dependent increase in nasal resistance (Fig. 4A). Moreover, the AUC(1 – 18 Hz) at 30 min for the ovalbumin (1% and 3%) were 1.77 F 0.18 and 3.18 F 0.72 cm H2Ol/ minHz and were significantly different from control animals,
Fig. 4. Area under complete frequency (1 – 18 Hz) curve analysis. (A) AUC for controls (n = 10) and ovalbumin-challenged (n = 10) animals. (B) AUC for controls and ovalbumin-challenged animals treated with chlorpheniramine (1 mg/kg iv, n = 8) and phenylpropanolamine (3 mg/kg iv, n = 8) at baseline and 30 min after ovalbumin nasal challenge. Each bar represents the mean F S.E.M. * P < .05 compared to control animals; * * P < .05 compared to vehicle-treated animals.
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0.78 F 0.18 cm H2Ol/minHz. The AUC(1 – 18 Hz) analysis also shows that chlorpheniramine (1 mg/kg iv), but not phenylpropanolamine (3 mg/kg iv), blocked allergic-induced nasal obstruction (Fig. 4B).
4. Discussion The forced oscillation method permits measurements of respiratory system impedance and its components as a function of frequency. The method has been used to study pulmonary mechanics in animals (Clercx, Gustin, Landser, & Van de Woestijne, 1993; Petak, Hall, & Sly, 1998; Preuss et al., 1999) and man (Delacourt et al., 2000; Farre, Peslin, Rotger, Barbera, & Navajas, 1999; Hall, Hantos, Wildhaber, Petak, & Sly, 2001). Moreover, the results generated by the forced oscillation technique are consistent with data generated by other methods used to evaluate pulmonary obstruction and bronchial reactivity (Farre et al., 1999). Only a few reports have described the use of the forced oscillation technique in human nasal studies (Aksamit et al., 1991; Fulton et al., 1984; Lorino et al., 1998). To date, the utility of the forced oscillation technique in animal nasal studies has not been discussed. In the present investigation, we studied the increases in nasal airway resistance produced by intranasally administered ovalbumin to sensitized guinea pigs using a modified rhinometric method based on that described by Salem and Clemente (1972). In addition, we evaluated nasal patency using the forced oscillation method. Interesting, baseline guinea pig nasal resistance values have not been extensively reported. Nevertheless, we found, using the modified rhinometric method, that our baseline nasal resistance values (7.9 F 0.9 cm H2Ol/min) for control animals were less than the conscious guinea pig nasal baseline resistance values (15 F 1.7 cm H2Ol/min) reported by Fujita et al. (1999). It must be pointed out that Fujita et al. used a plethysmographic technique to study nasal patency. Consequently, measurements of nasal resistance were not exclusively limited to an evaluation of upper-airway patency but also incorporated an assessment of pulmonary resistance. Curran, O’Halloran, and Bradford (1998) on the other hand, used a rhinometric-based technique to study the effects of cold air on nasal resistance in anesthetized guinea pigs. These investigators reported baseline nasal resistance values of approximately 0.21 cm H2Ol/min in this species. Therefore, it appears that baseline nasal resistances in the guinea pig may vary significantly depending on the method utilized to determine this respiratory parameter. In fact, we found that fundamental differences in the delivery of air (and airflow rates) to the nasal cavity between our modified rhinometric method and the forced oscillation method did not allow us to directly compare baseline nasal resistance values obtained from each method. For example, using the rhinometric method, a cannula was placed into the esophagus and positioned next to the nasopharynx. This positioning allowed retrograde airflow across the nasal cavity. In
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contrast, in the forced oscillation method, air was delivered orthograde through a nasal cannula and resistance was determined across one nares. Nevertheless, we found that the qualitative results from each of the methods were not different. For example, the increase in nasal resistance produced by 3% ovalbumin at 30 min posttreatment was 99 F 14% and 91 F 14% for the rhinometric and the forced oscillation methods, respectively. The forced oscillation technique permits the measurement of airway mechanics over a wide-frequency range. The data can then be fitted to theoretical models of the respiratory system to identify which elements of the airway are responsible for the changes in mechanics. Many models of the respiratory system have been proposed, but the constant phase model of Petak, Hantos, Adamicza, Asztalos, and Sly (1997) fits experimental data well over a range of physiological and pathological states with a small number of model elements. In contrast, there are no models of the nasal system to fit to forced oscillation data and we did not attempt such an analysis. However, the relatively flat resistance –frequency graph (Fig. 3) suggests that there is not much additional information to be gained by making measurements of resistance over the frequency range 1 –18 Hz as compared to a single-spot frequency. This is borne out by the similarities between Figs. 2B and 4B. We found that the histamine H1 antagonist chlorpheniramine blocked the nasal obstruction produced by ovalbumin in sensitized guinea pigs. The nasal activity of chlorpheniramine was demonstrated using both the modified rhinometric method and the forced oscillation technique. A second H1 antagonist tested, pyrilamine, was also found to block the obstruction produced by nasal exposure to ovalbumin challenge. Our results with chlorpheniramine and pyrilamine are consistent with previous studies showing that H1 antagonists block nasal obstruction in allergic guinea pigs (Narita & Asakura, 1993; Ohkawa, Ukai, Miyahara, Takeuchi, & Sakakura, 1999). In contrast to the effects of chlorpheniramine and pyrilamine, we found that phenylpropanolamine did not block nasal obstruction. Our results with phenylpropanolamine are not consistent with the findings of Imai et al. (2001), denoting that a-agonists produce nasal decongestion in the guinea pig. Nevertheless, interpretation of our finding suggests that nasal vasodilatation does not appear to significantly contribute to allergic nasal obstruction in the guinea pig. Consequently, we propose that nasal obstruction in the allergic guinea pig may be distinct from nasal congestion in man. Unquestionably, a-adrenergic agonist decongestants ameliorate nasal vascular engorgement characteristic of human nasal congestion. Furthermore, a-agonist decongestants also effectively improve nasal patency in the cat, dog, and pig (Koss, Yu, Hey, & McLeod, 2002a,b; Lacroix, 1989; McLeod, Mingo, Herczku, DeGennaro-Culver, et al., 1999; McLeod, Mingo, Herczku, Corboz, et al., 1999). Allergy-induced nasal obstruction in the guinea pig is likely due to a histamine H1 receptor-mediated increase in nasal vascular permeability
and an increase in glandular secretions (Gawin, Baraniuk, & Kaliner, 1992). A second distinction between guinea pig nasal obstruction and human nasal congestion is that in man, H1 antagonists are not very effective decongestants (Spector, 1999). The differences in the nasal pharmacological effects of the H1 antagonists and the a-decongestants in the guinea pig and man may be related to differences in the morphological organization of upper airways among the two species. In support of this hypothesis, using conventional light and electron microscopy, Pastor, Amores, Villaverde, Calvo, and Spreklsen (1990) identified two distinct nasal conchae in the guinea pig, namely, the nasal turbinate and the maxilloturbinate. In humans, typically three turbinates (inferior, middle, and superior) are found. In addition, Pastor et al. also found that the anterior sections of guinea pig turbinates, like human turbinates, are highly vascularized. However, in contrast to findings in humans, the anterior portion of the guinea pig turbinate does not contain glands. Nasal glands in the guinea pig nasal cavity are distributed in more posterior sections of the nasal turbinates. Another significant distinction between guinea pigs and humans relates to the functionality of the upper respiratory system. For example, it should be pointed out that humans and most mammals have the capacity to breathe through their nose as well as their mouth. On the other hand, guinea pigs and most rodents are nasal-obligatory breathers. The extent to which these distinct morphological and functional differences in the upper respiratory airways of guinea pigs relative to humans affect nasal physiology remains to be determined. Nevertheless, a consideration of the unique features of the guinea pig nose should be considered when this species is used to extrapolate nasal physiology and pharmacology to humans. In summary, we characterized the nasal-decongestant effects of H1 antagonists and the a-adrenergic agonist, phenylpropanolamine, using two different methods in the guinea pig. In both of these models, the H1 antagonists significantly blocked ovalbumin-induced nasal obstruction; however, the a-agonist produced little or no effect on allergic nasal obstruction. In light of the present findings, we suggest that additional studies to fully characterize the nasal effects of a-adrenergic agonists in the guinea pig are warranted. Furthermore, it is important to recognize the utility of the forced oscillation method as an effective technique in evaluating nasal patency in preclinical animal models. References Aksamit, T., Duggan, C., Watson, A., & Pride, N. D. (1991). Use of oscillation methods to measure nasal airflow resistance. European Respiratory Review, 1, 232 – 235. Albert, D. H., Malo, P. E., Tapang, P., Shaughnessy, T. K., Morgan, D. W., Wegner, C. D., Curtin, M. L., Sheppard, G. S., Xu, L., Davidsen, S. K., Summers, J. B., & Carter, G. W. (1998). The role of platelet-activating factor (PAF) and the efficacy of ABT-491, a highly potent and selective PAF antagonist, in experimental allergic rhinitis. Journal of Pharmacology and Experimental Therapeutics, 284, 83 – 88.
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