### 94:1563-1571, 2003. First published Nov 27, 2002; doi:10.1152/japplphysiol.00572.2002 J Appl Physiol 

## M. Cloutier and Andrea K. Hubbard John B. Morris, Peter T. Symanowicz, Joshua E. Olsen, Roger S. Thrall, Michelle

You might find this additional information useful...

45 articles, 16 of which you can access free at: This article cites http://jap.physiology.org/cgi/content/full/94/4/1563#BIBL

8 other HighWire hosted articles, the first 5 are: This article has been cited by

[PDF] [Full Text] [Abstract] , September 1, 2006; 291 (3): L426-L435. Am J Physiol Lung Cell Mol Physiol D. B. T. Anh, P. Faisca and D. J.-M. Desmecht mouse strains Differential resistance/susceptibility patterns to pneumovirus infection among inbred [PDF] [Full Text] [Abstract] , October 1, 2006; 93 (2): 411-421. Toxicol. Sci. R. P. Vaughan, M. T. Szewczyk Jr, M. J. Lanosa, C. R. DeSesa, G. Gianutsos and J. B. Morris Irritation Response to Inspired Irritant Vapors Adenosine Sensory Transduction Pathways Contribute to Activation of the Sensory [PDF] [Full Text] [Abstract] , July 1, 2008; 104 (1): 198-209. Toxicol. Sci. C. R. DeSesa, R. P. Vaughan, M. J. Lanosa, K. G. Fontaine and J. B. Morris Capsaicin Sulfur-Containing Malodorant Vapors Enhance Responsiveness to the Sensory Irritant [PDF] [Abstract] , August 1, 2008; 24 (7): 447-490. Toxicology and Industrial Health O Faroon, N Roney, J Taylor, A Ashizawa, M. Lumpkin and D. Plewak Acrolein health effects [PDF] [Full Text] [Abstract] , June 15, 2009; 182 (12): 7430-7439. J. Immunol. J. M. Cyphert, M. Kovarova, I. C. Allen, J. M. Hartney, D. L. Murphy, J. Wess and B. H. Koller Bronchoconstriction Cooperation between Mast Cells and Neurons Is Essential for Antigen-Mediated

including high-resolution figures, can be found at: Updated information and services http://jap.physiology.org/cgi/content/full/94/4/1563

can be found at: Journal of Applied Physiology about Additional material and information http://www.the-aps.org/publications/jappl

This information is current as of September 21, 2009 .

http://www.the-aps.org/. ISSN: 8750-7587, ESSN: 1522-1601. Visit our website at Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society. those papers emphasizing adaptive and integrative mechanisms. It is published 12 times a year (monthly) by the American publishes original papers that deal with diverse areas of research in applied physiology, especially Journal of Applied Physiologyon September 21, 2009jap.physiology.orgDownloaded from

# Immediate sensory nerve-mediated respiratory responses to irritants in healthy and allergic airway-diseased mice

John B. Morris, 1 

### Peter T. Symanowicz, 1 

### Joshua E. Olsen, 1 

### Roger S. Thrall, 2 

### Michelle M. Cloutier, 3 

### and Andrea K. Hubbard 1

1 University of Connecticut Pulmonary Research Consortium, Department of Pharmaceutical Sciences, University of Connecticut, Storrs 06269; and Departments of 2Medicine and

3Pediatrics, University of Connecticut Health Center, Farmington, Connecticut 06032

Submitted 1 July 2002; accepted in final form 25 November 2002

Morris, John B., Peter T. Symanowicz, Joshua E.Olsen, Roger S. Thrall, Michelle M. Cloutier, and Andrea K. Hubbard. Immediate sensory nerve-mediated respiratory responses to irritants in healthy and allergic airway-diseased mice. J Appl Physiol 94: 1563–1571, 2003. First published November 27, 2002; 10.1152/japplphysiol.00572.2002.—The immediate responses of the upper respiratory tract (URT) to the irritants acrolein and acetic acid were examined in healthy and allergic airway-diseased C57Bl/6J mice. Acrolein (1.1 ppm) and acetic acid (330 ppm) vaporsinduced an immediate increase in flow resistance, as measured in the surgically isolated URT of urethane-anesthetized healthy animals. Acrolein, but not acetic acid, induced a small URT vasodilatory response. In awake spontaneously breathing mice, both vapors induced a prolonged pause at the start of expiration (a response mediated via stimulation of nasal trigeminal nerves) and an increase in total respiratory specific airway flow resistance, the magnitude of which was similar to that observed in the isolated URT. Both responses were significantly reduced in animals pretreated with large doses of capsaicin to defunctionalize sensory nerves, strongly suggesting a role for sensory nerves in development of theseresponses. The breathing pattern and/or obstructive responses were enhanced in mice with ovalbumin-induced allergic airway disease. These results suggest that the primary responses to acrolein and acetic acid vapors are altered breathing patterns and airway obstruction, that sensory nerves play an important role in these responses, and that these responses are enhanced in animals with allergic airway disease.
acrolein; acetic acid; ovalbumin; upper respiratory tract; specific airway resistance

THE RESPIRATORY TRACT IS INNERVATED 

### with a rich supply of sensory nerves that detect and initiate responses to inspired chemicals (1a, 30). The sensory nerve network consists of thick neurofilament expressing myelinated A -fibers and thin nonmyelinated C fibers. It is believed the A -fibers serve primarily a mechanosensoryfunction, whereas C fibers are important in the detection of irritant chemicals (15, 30, 44). The distinction isnot complete, however, because these functions overlap. The best-characterized sensory nerve stimulant is the plant toxin capsaicin, which acts via stimulation of the vanilloid receptor. At low doses, this toxin produces acute transient stimulation; at high doses, it causes a prolonged degeneration of sensory nerves, presumably through an excitotoxic mechanism (14, 18, 43, 48). Capsaicin exerts its effects preferentially on C fibers, which express high levels of the vanilloid receptor. However, a subset of A -fibers may be responsive to this toxin as well (20, 23, 36, 48). C fibers can be separated into two types on the basis of lectin binding: IB4



### and IB4  

### fibers. The former express large amounts of neuropeptides, including substance P and calcitonin gene-related peptide; the latter do not (40). Stimulation of respiratory sensory nerves produces centrally mediated afferent reflex responses and local tissue responses (1a, 4–7, 15, 30). The afferent reflexresponses include cough and changes in breathing patterns. By definition, “sensory irritants” are compounds that stimulate nasal trigeminal C fibers and decrease breathing rate by inducing a prolonged pause at the start of expiration (for reviews of sensory irritation mechanisms, see Refs. 1a, 9, and 30). This reflex change is blocked by trigeminal nerve ligation and reproduced by trigeminal ganglion stimulation (1a). The sensory irritation response is particularly marked in mice. The potency of sensory irritants is quantified by the concentration that produces a 50% decrease in breathing rate (RD 50 

### ) (1a). Capsaicin is a potent sen

sory irritant (1a, 30); in our hands a 50% reduction inbreathing frequency in the mouse is induced by exposure to capsaicin at

 0.2  g/l.Stimulation of C fibers can also cause local responses, including vasodilation, neurogenic edema, mucus secretion, and/or airway obstruction, which aremediated, in part, by release of neuropeptides via antidromal stimulation (4–7, 32). Presumably the neuropeptide-rich IB4



### fibers (40) are most important inthese responses. Activation of parasympathetic efferents may also play a role in the mucus secretory and airway obstructive responses (4, 5).

Previous studies in our laboratory were aimed at characterizing upper respiratory tract (URT) sensory

Address for reprint requests and other correspondence: J. B. Morris,Dept. of Pharmaceutical Sciences, Box U-2092, University of Connecticut, Storrs, CT 06269 (E-mail: morris@uconnvm.uconn.edu). 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. J Appl Physiol 94: 1563–1571, 2003. First published November 27, 2002; 10.1152/japplphysiol.00572.2002.

8750-7587/03 $5.00 Copyright © 2003 the American Physiological Society http://www.jap.org 1563on September 21, 2009jap.physiology.orgDownloaded from

### nerve-mediated responses to inspired irritants in the rat and included measures of afferent re fl ex responses(changes in breathing pattern) and local tissue responses, including neurogenic edema, airway obstruction, and mucus secretion (26, 28, 39). (The URT is de fi ned as all portions of the respiratory tract anteriorto and including the larynx.) These studies have examined the effects of two reactive electrophilic vapors, acrolein and ethyl acrylate, and two vapors for which tissue acidi fi cation is believed to be important, aceticacid vapor and acetaldehyde. [Acetaldehyde is metabolized in nasal tissues to acetic acid via aldehyde dehydrogenase, and this is believed to be a critical step inits nasal toxicity (24, 38, 39).] Of the responses measured, the most sensitive for all four vapors was vasodilation. The vasodilatory response to every vapor was greatly reduced and/or abolished in animals pretreated with capsaicin, confi rming a key role for sensory nerves. The precise mediators involved in this response are not known; however, preliminary studies suggest a role for calcitonin gene-related peptide and nitric oxide (27

– 29).

The aims of the present study were twofold: 1 ) to characterize the response of the mouse nose to acrolein and acetic acid vapor to allow for comparison with our laboratory’ s earlier rat studies (26, 28, 29, 39) and 2 ) tocompare responses in healthy and allergic airway-diseased mice. The initial experiments relied on methodologies identical to those used in our previous rat studies to facilitate comparisons. Toward these ends,the URT of the urethane-anesthetized mouse was isolated by tracheostomy and insertion of an endotrachealtube. The mouse was then placed in a nose-only inhalation chamber, and irritant-laden air was drawn continuously through the isolated URT at a constant fl owrate (25 ml/min) for 50 min. Chamber air also contained acetone vapor. Uptake of acetone vapor wasmonitored throughout exposure to provide a continuous measure of URT perfusion rates (11, 28, 29, 35). The pressure drop across the URT was also measured during exposure to assess airway fl ow resistance throughout the exposure. Before exposure, animals were injected with Evans blue, a dye that binds to albumin. Dye content in nasal tissues and nasal airway lavage fl uid (obtained immediately after exposure) provides a measure of plasma protein leakage/secretion into these compartments (28, 29, 31, 39). The same technique was used to quantitate URT uptake ef fi

ciency of acrolein and acetic acid vapors. Initial studies on the mouse isolated URT revealed that the most sensitive response to acrolein and acetic acid vapors was increased fl ow resistance, rather than vasodilation, as observed in the rat. Because airwayobstruction can be measured noninvasively by plethysmography, subsequent studies were performed to assess irritant-induced airway obstruction and breathing pattern changes in intact spontaneously breathing mice. The role for sensory nerves in the responses wasexamined by pretreatment with capsaicin to defunctionalize sensory nerves (18, 43). In addition, the effects of atropine were examined to provide information on the potential role of cholinergic mechanisms. Somestudies have suggested that humans with allergic airway disease, i.e., rhinitis and/or asthma, may have heightened sensitivity to inspired irritants (33, 46).Indeed, subjects with asthma develop an airway obstructive response to sulfuric acid aerosol at concentrations to which healthy subjects are unresponsive (46).Therefore, in mice with ovalbumin (OVA)-induced allergic airway disease, the effects of acetic acid and acrolein were also examined using the model developed previously in our laboratories (50) to determine whether a similar phenomenon could be demonstrated in this animal model.

METHODS

Animals and chemicals. Male or female C57Bl/6J mice (Jackson Laboratories, Bar Harbor, ME) were housed over hardwood bedding in animal rooms maintained at 22

– 25 ° Cwith a 12:12-h light-dark cycle (lights on at 6:30 AM). Animals weighed 18

– 25 g and were 10

– 18 wk of age. Food (Purina Laboratory Chow) and tap water were provided ad libitum. Animals were acclimated for  2 wk before use. All chemicals were obtained from Sigma Aldrich (St. Louis, MO) unless otherwise indicated.

Irritant atmosphere generation and analysis. Chamber atmospheres were generated with a syringe-pump system. Aqueous solutions of acrolein or acetic acid and/or acetone (Fisher Scienti fi c, Springfi eld, NJ) were prepared daily and fed into a heated tube maintained at 60 ° C. Air (5 l/min) was passed through the tube and into the exposure chamber. The generation system was operated for  45 min before use toallow for equilibration. Chamber concentrations were controlled by changing the irritant concentrations in the aqueous generation fl uids. Isolated URT exposure took place in a nose-only chamber. Exposures to intact mice took place in the Buxco double plethysmograph (see below).

Air samples were drawn from the nose-only chamber orplethysmograph during exposure to determine exposure concentration using methods employed in our laboratory’ s previous studies (28, 29, 39). For acrolein or acetone analysis, air was drawn through a gas-sampling valve attached to a gas chromatograph (model 3600, Varian, Sugar Land, TX) with fl ame ionization detection. A 15-m DB-WAX column (J & W Scienti fi c, Folsom, CA) was used with a column temperature of 33 ° C and a carrier gas (N 2 ) fl ow rate of 30 ml/min. To measure acetic acid concentration, chamber air was drawn through two impingers in series, each containing 10 ml of distilled water (100 ml/min fl ow rate). The concentration of acetate in each impinger was then determined via HPLC with ultraviolet detection at 210 nm (model 2510, Varian). (The second impinger collected 20-fold less acetic acid than the fi rst, indicating the high collection ef fi ciency of the system.) Analysis was performed using a mobile phase consisting of 5:95 vol/vol acetonitrile-0.1% H 3 PO 4 at a fl ow rate of 1.3 ml/min. The retention time for the acetate peak

was  2.1 min.

Isolated URT exposures. Initial studies focused on characterizing the responses of the isolated URT and included measures of perfusion rate (as assessed by acetone vapor uptake), fl ow resistance, plasma protein extravasation (tissue Evans blue dye content), and mucus secretion (lavage Evans blue dye content). Control mice were exposed to clean air. Nominal exposure concentrations for acrolein and acetic acid were 1.2 and 300 ppm, respectively. These exposure concentrations were estimated to approximate the RD 50 

### 1564 NASAL REFLEX RESPONSES TO ACROLEIN AND ACETIC ACID

J Appl Physiol • VOL 94 • APRIL 2003 • www.jap.orgon September 21, 2009jap.physiology.orgDownloaded frombased on pilot studies. Our laboratory’ s previous studies on the rat indicated that local tissue responses were easily demonstrable at RD 50 of each irritant (26, 28, 29, 39).

The experimental methodology has been described in detail (28, 29). Mice were anesthetized with urethane (1.3 g/kg ip). After the onset of anesthesia, the trachea was isolated and incised and an endotracheal tube was inserted in a cephalad direction until its tip lay at the larynx. Evans blue dye (30 mg/kg) was injected into the tail vein. The animalwas then placed in the nose-only chamber in a supine position, and chamber air was drawn through the isolated URT at a constant fl ow rate of 25 ml/min for 50 min, which was the highest fl ow rate that could be easily maintained. The tracheotomized animals respired room air during the exposure;thus only the URT was directly exposed to the irritant vapors. Chamber temperature was maintained at

 39 ° C, and water content was  33 mg/l, corresponding to  75% relative humidity at 37 ° C. These conditions were necessary to prevent nasal dehydration. The chamber walls were heated toprevent condensation. Immediately after exposure, the animal was removed and killed via exsanguination by incision of the abdominal aorta. The URT was then lavaged with 1 ml of saline (administered into the endotracheal tube and collected at the external nares). The lavage fl uid was diluted 1:10 vol/vol with formamide. The vasculature was perfused with 10 ml of saline, the animal was decapitated, the skull was split sagittally, and nasal tissues were collected and digested in 3 ml of formamide. The Evans blue dye content of the lavage and tissue was determined fl uorometrically as described previously (28, 31).

URT acetone vapor uptake was monitored during exposure as a measure of URT perfusion rates by the methods used in our laboratory’ s previous studies (26, 28, 31). Brie fly, vapor concentrations in chamber air (C in ) and in air that had been drawn through the isolated URT (C
ex ) were measured by gas chromatography as described above. C in was measured im

mediately before and immediately after the 50-min exposure. The ratio of the before and after samples averaged 99.9 4.3%, indicating that exposure concentration remained constant. The gas-sampling valve injected 0.5-ml samples intothe gas chromatograph at 3-min intervals to provide continuous monitoring of C
ex during exposure. Acetone uptake rates were calculated from the ratio of C
ex to C in and nor

malized to an inspired concentration of 44 ppm (100  g/l).

URT fl ow resistance was also monitored throughout exposure. Toward this end, the endotracheal tube contained a “ T, ” one side of which was connected to a differential pressuretransducer (model DP45, Validyne, Northridge, CA) for measurement of URT pressure drop at 5-min intervals. Flow resistance was obtained by dividing the pressure drop by the fl ow rate (25 ml/min). In a separate group of mice, URT uptake ef fi ciencies of acrolein and acetic acid vapors were measured. Animals were exposed as described above, except acetone vapor was not present. Irritant-laden air was drawn through the URT for 50 min ( fl ow rate 25 ml/min), and irritant concentration was measured in the URT exiting air (C
ex ). Acrolein concentra

tion was measured in the URT exiting air by gas chromatography (see above). For acetic acid, URT exiting air was drawn through two midget impingers in series, each containing 5 ml of distilled water, and the impinger fl uid was analyzed for acetic acid content by HPLC, as described above. The uptake ef

fi ciency was calculated from the inspired (chamber) air concentration (C in ) and the URT exiting air concentration (C
ex ) and expressed as a percentage. This methodology has been described in detail (25). Effect of pharmacological pretreatment. The next experiment was aimed at determining the effects of capsaicin or atropine pretreatment on the responses to acrolein and acetic acid vapors. Nominal exposure concentrations were 1.2 and300 ppm, respectively. In initial studies, capsaicin was administered at the dose used in our previous rat studies (50 mg/kg). This dose was lethal in the mouse. Therefore, it was decided to split the capsaicin dose and to perform a capsaicin dose-response study. All capsaicin-treated mice received the toxin at 25 mg/kg on day 1, and on day 2 the animals received the toxin at 25, 50, or 75 mg/kg. For treatment, animals were fi rst anesthetized with tribromoethanol (Avertin) and thentreated with theophylline (10 mg/ml sc in 10:90 vol/vol ethanol-distilled water) and terbutaline (0.1 mg/ml ip in saline). Animals then received capsaicin (5 mg/ml sc in 1:1:8 vol/vol/ vol ethanol-Tween 80-saline). Control mice were injected with drug and capsaicin vehicle. Atropine (1.25 mg/ml in saline) was administered at 5 mg/kg ip 30 min before irritant exposure. (In our hands, this dose inhibited the response to inspired methacholine aerosol.) Control mice were injected with saline. The isolated URT studies suggested that upper airway obstruction was the most sensitive response to the irritants.Because airway obstruction can be monitored in intact spontaneously breathing mice by plethysmography, this approach was selected for this study relying on standard protocols (1a, 2, 9). Respiratory responses were measured in a doubleplethysmograph (Buxco, Sharon, CT) using the Buxco noninvasive airway mechanics software. Animals were restrained in the double plethysmograph but were not anesthetized. Irritant-laden air was drawn from the nose-only exposureand through the head-space side of the double plethysmograph at a fl ow rate of 0.4 l/min. Three responses were measured: breathing rate, expiratory pause duration, and speci fi c airway resistance (sRaw). After a  10-min acclimatization period, animals were exposed to clean air for a baseline 10-min period and then to irritant for 10 min. Breathing parameters were collected during the baseline and exposure periods. One-minute average values were recorded,and the peak responses (1-min averages) were used for statistical analysis. Breathing frequency was expressed as percentage of baseline according to standard protocols (2). Absolute values for expiratory pause duration (ms) and sRaw (cmH 2 O

 s) were used.

Effect of OVA-induced allergic airway disease. To examine the responses of mice with allergic airway disease, the OVA treatment protocol of Yiamouyiannis et al. (50) was used.This model has been extensively characterized in our previous studies (34, 49, 50). Brie fly, animals received three weekly intraperitoneal injections of 25  g of OVA (grade V), adsorbed to 2 mg of aluminum hydroxide. One week after the last injection, animals were exposed for 1 h/day to aerosolized OVA. Exposures took place in a directed-airfl ow nose-only inhalation chamber (CH Technologies). Atmospheres weregenerated by nebulization (Lovelace Nebulizer, In-Tox Products, Albuquerque, NM) of 1% OVA in saline. Aerosol concentration averaged

 20 mg/m 3 (

 1.6-  m mass median aerodynamic diameter). Control animals received no OVA exposures. Chamber temperatures were 22

– 25 ° C, and relative humidity was 20

– 40%. To assess irritant sensitivity, animals were exposed to irritant for 10 min with continuous monitoring of breathingparameters by a protocol identical to that used in the capsaicin studies. In each animal, irritant sensitivity was examined 24 h after the last OVA aerosol exposure. Two timepoints in the OVA model were selected. Animals were examined 24 h after the third daily OVA aerosol challenge expo

### 1565 NASAL REFLEX RESPONSES TO ACROLEIN AND ACETIC ACID

J Appl Physiol • VOL 94 • APRIL 2003 • www.jap.orgon September 21, 2009jap.physiology.orgDownloaded fromsure (OVA-d3) to examine a time when the airway infl ammation is in its early stages. Another group of animals was examined 24 h after the 7th

– 10th daily OVA aerosol challenge exposure (OVA-d7

– 10) to examine a time when the infl ammation was fully developed (34, 50). As in the capsaicin studies, the peak response (1-min averages) during the exposure period was used for statistical analysis.

Statistical analysis. Values are means  SD and werecompared among groups by one- or two-factor ANOVA followed by Newman-Keuls test. Animal groups consisted ofthree to eight animals. Repeated-measures ANOVA was performed on the URT uptake and resistance data. RD 50 values were calculated by logarithmic linear regression of breathing frequency vs. exposure concentration data (1a). P  0.05 was required for signi fi cance. Statistical calculations were performed with Statistica software (Stat Soft, Tulsa, OK).

RESULTS



### Isolated URT studies. Measured exposure concentrations averaged 1.1 and 330 ppm for acrolein and aceticacid, respectively. The effects of the irritants on acetone vapor uptake are shown in Fig. 1. Repeatedmeasures ANOVA revealed that acrolein produced a signi fi cant increase in acetone uptake rate (P  0.05) over the levels in air-exposed control mice. An effect of exposure time (the repeated measure) was not detected (P  0.05). Uptake rates were increased from

 4 to 5

 g/min by this irritant. Although a transient increasemay have occurred, acetic acid was without statistically signi fi cant effect on uptake. URT fl ow resistance during exposure to these irritants is shown in Fig. 2. Both vapors produced a rapid and statistically signi fi

cant increase in URT fl ow resistance (P  0.05, repeated-measures ANOVA). An effect of exposure time was not detected (P  0.05), suggesting that this was an immediate and sustained response to the irritants. Flow resistance increased from control values of

 2 to

 5 cmH 2 

### O

 ml

1



###  s. Nasal tissue and nasal lavage Evans blue dye content in control and irritant-exposed animals are shown in Fig. 3. Values were similar in control and exposed animals (P  0.05, ANOVA), suggesting that the irritants did not induce signi fi cant plasma protein extravasation and/or mucus secretion during exposure.

The URT uptake ef fi ciency studies revealed thatacrolein and acetic acid were scrubbed from the airstream with high ef fi ciency in the URT. Acrolein exposure concentration (C in 

### ) averaged 1.2 ppm in this study; acrolein vapor was not detected in URT exiting

Fig. 1. Upper respiratory tract (URT) acetone uptake rates during exposure to clean air (control), 1.1 ppm acrolein, or 330 ppm acetic acid. SD bars are omitted for the sake of clarity; SD averaged

 0.5

 g/min. Acrolein and acetic acid data are displaced slightly to the leftand right, respectively, for clarity. Repeated-measures ANOVA revealed no statistical effect of exposure time, a signi fi cant difference between exposure groups (P  0.05), and no statistical interaction between exposure group and time. Newman-Keuls test revealed that acetone uptake in the acrolein, but not the acetic acid, group was signi fi cantly elevated over control levels. Each group consisted of 9

– 12 animals. Fig. 2. URT fl ow resistance (mean  SD) during exposure to clean air (control), 1.1 ppm acrolein, or 330 ppm acetic acid. Acrolein andacetic acid data are displaced slightly to the left and right, respectively, for clarity. Repeated-measures ANOVA revealed no statistical effect of exposure time, a signi fi cant difference between exposure groups (P  0.05), and no statistical interaction between exposure group and time. Newman-Keuls test revealed that fl ow resistance in the acrolein and acetic acid groups was signi fi cantly elevated over control levels (P  0.05). Each group consisted of 9

– 12 animals.

Fig. 3. Evans blue dye content in nasal tissue (  g) and nasal lavage (  g/ml) in control (air-exposed) mice and mice exposed to 1.1 ppm acrolein and 330 ppm acetic acid. Values are means  SD. ANOVA detected no signi fi cant differences among exposure groups. Each group consisted of 9

– 12 animals. 

### 1566 NASAL REFLEX RESPONSES TO ACROLEIN AND ACETIC ACID

J Appl Physiol • VOL 94 • APRIL 2003 • www.jap.orgon September 21, 2009jap.physiology.orgDownloaded from

### air. On the basis of the limit of detection of the gas chromatographic technique of 0.1 ppm, URT uptake ef

fi ciencies were estimated to be  92%. Acetic acid exposure concentration averaged 285 ppm in this study; the concentration in URT exiting air averaged 9  10 ppm; URT uptake ef fi ciency averaged 97  4%. Pharmacological pretreatment studies. Exposure concentrations for acrolein and acetic acid were similar to those in the isolated URT study, averaging 1.3 and 290 ppm, respectively. There were no indications of sneezing or water rhinorrhea during exposure, nor did the animals demonstrate avoidance behavior. Baseline breathing frequency, expiratory pause duration, and sRaw averaged 301  23 breaths/min, 9.6  2.0 ms, 2.5  0.23 cmH 2 

### O

 s, respectively. Both vapors caused a signi fi cant decrease in respiratory rate, a signi fi cant expiratory pause, and a signi fi cant increase in sRaw (Table 1). The increases in respiratory resistance in absolute terms (cmH 2 

### O

 ml

1



###  s) are not known, inas

much as the plethysomgraphic technique provides a measure of sRaw, not true resistance. With the use of an estimated functional residual capacity for the mouse of 0.5 ml, the increase in resistance induced by acrolein and acetic acid is estimated to be 1.2 and 4.2 cmH 2 

### O

 ml

1



###  s, respectively. This should be viewed as a rough estimate, because it is not known whether either irritant altered functional residual capacity. However, these values are similar to the fl ow resistance changes observed in the isolated URT (Fig. 2).For each response measure (respiratory rate, expiratory pause duration, and increase in sRaw; Table 1), data were analyzed by two-factor ANOVA, with the factors being irritant (acrolein vs. acetic acid) and capsaicin dose (25  25, 25  50, and 25  75 mg/kg).Regardless of the measure, the two-factor ANOVA revealed a signi fi cant difference between vapors, with acetic acid producing larger responses, and a signi fi

cant effect of capsaicin pretreatment, with signi fi cantly lower responses in the 20  50 and 20  75 groups than in the vehicle control or 25  25 group (NewmanKeuls test). A signi fi cant interaction between irritant and capsaicin dose was not detected, indicating that capsaicin exerted statistically similar effects on the responses to both irritants.

Atropine was without statistically signi fi cant effect (P  0.05, ANOVA) on the responses to 1.4 ppm acrolein or 260 ppm acetic acid. The respiratory rate, expiratory pause, and increase in sRaw during acetic acid exposure averaged 60  12% of baseline, 224  91 ms, and 0.82  0.18 cmH 2 

### O

 s, respectively, in vehicletreated mice compared with 55  7% of baseline, 244  47 ms, and 0.92  0.78 cmH 2 

### O

 s, respectively, in atropine-pretreated mice. For acrolein, these values averaged 58  18% of baseline, 215  124 ms, and 1.48  0.82 cmH 2 

### O

 s, respectively, in vehicle-treated mice compared with 49  4% of baseline, 268  52 ms, and 1.00  0.86 cmH 2 

### O

 s, respectively, in atropinepretreated mice.Concentration response in healthy and allergic airway-diseased mice. The effect of acrolein exposure onbreathing frequency in naive and OVA-challenged animals is shown in Fig. 4. Acrolein exposure concentrations were 0.3, 1.6, and 3.9 ppm. A pause during expiration was observed in the irritant-exposed animals. Two-factor ANOVA revealed a signi fi cant difference between exposure concentrations (P  0.001), a significant difference between OVA groups (P  0.01), and no interaction between factors. Newman-Keuls test revealed that the response in the naive and OVA-d3animals were statistically similar and that the response was greater in the OVA-d7

– 10 than in the

naı  ̈ve or OVA-d3 animals. RD 50 

### values were 1.59, 1.44, and 0.82 ppm in naive, OVA-d3, and OVA-d7

– 10 animals, respectively (log-linear regression). Acrolein also induced an increase in sRaw, with the increase over baseline averaging 0.29  0.08, 2.03  0.21, and 1.95 

Table 1. Respiratory tract responses to acrolein and acetic acid vapors in normal and capsaicin-pretreated mice

Vehicle Capsaicin

25  25 mg/kg 25  50 mg/kg 25  75 mg/kg
Respiratory rate, %baseline

Acrolein 55  4 63  13 77  15* † 85  11* † Acetic acid 47  6 53  9 71  14* † 76  6* † Expiratory pause, ms

Acrolein 202  66 155  71* 64  94* † 11  9* † Acetic acid 321  39 250  38* 159  33* † 119  27* † sRaw, cmH 2 O

 s

Acrolein 0.56  0.34 0.58  0.48 0.13  0.23* 0.09  0.15* † Acetic acid 2.06  0.70 1.47  0.55 1.08  0.29* 0.89  0.15* †

Values are means  SD of 4

– 6 mice. Expiratory pause and speci fi c airway resistance (sRaw) are expressed as increase over baseline. Animals were exposed in a double plethysmograph to 1.3 ppm acrolein or 290 ppm acetic acid, and responses were monitored during the 10-min exposure. Animals were pretreated on 2 consecutive days with vehicle or capsaicin at 25 mg/kg followed by 25 mg/kg, 25 mg/kg followed by 50 mg/kg, or 25 mg/kg followed by 75 mg/kg and exposed to acrolein and acetic acid vapor 1 and 2 wk later, respectively. Baseline values for respiratory rate, expiratory pause, and sRaw averaged 301  23 breaths/min, 9.6  2.0 ms, 2.5  0.23 cmH 2 O

 s, respectively. In vehicle-treated animals, respiratory rates were signi fi cantly  100% of baseline, and increases in expiratory pause duration and sRaw were signi fi cantly  0 (P  0.05, t -test). Data were analyzed by 2-factor ANOVA, with factors being irritant (acrolein or acetic acid) or capsaicin dose. A signi fi cant difference between irritants was detected for each measure (P  0.002 in all cases). A signi fi cant difference between capsaicin dose groups was also detected for each measure (P  0.02 in all cases). *Statistically different from vehicle; † statistically different from 25  25 mg/kg capsaicin (P  0.05, Newman-Keuls test). 

### 1567 NASAL REFLEX RESPONSES TO ACROLEIN AND ACETIC ACID

J Appl Physiol • VOL 94 • APRIL 2003 • www.jap.orgon September 21, 2009jap.physiology.orgDownloaded from

### 0.40 cmH 2 

### O

 s in the 0.3, 1.6, and 3.9 ppm exposure groups (P  0.05, ANOVA). (Baseline sRaw values were similar in naive and OVA groups.) The sRaw response observed in this study was greater than that observed in the capsaicin study (Table 1). The reasons are unclear. The sRaw response was not increased over that of naive animals in the OVA-d3 or OVA-d7

– 10 groups (P  0.05, 2-factor ANOVA). In the OVA-d7

– 10 group, the increase in sRaw averaged 0.5 and 1.9 cmH 2 

### O

 s at 0.3 and 1.6 ppm, respectively.

The effect of acetic acid vapor on breathing frequency in naive and OVA-treated animals is shown in Fig. 5. Acetic acid exposure concentrations averaged 82, 340, and 660 ppm. A pause during expiration was observed in the irritant-exposed animals. Two-factor ANOVA revealed a signi fi cant difference between exposure concentrations (P  0.001), a signi fi cant difference between OVA groups (P  0.01), and no signi fi cant interaction between factors. Newman-Keuls test revealed that the responses in the naive and OVA-d3 animals were statistically similar and that the response was greater in the OVA-d7

– 10 than in the naive or OVA-d3 animals. RD 50 

### values were 239, 247, and 136 ppm in naive, OVA-d3, and OVA-d7

– 10 animals, respectively (log-linear regression). Acetic acid vapor also induced airway obstruction, as indicated by a dose-dependent increase in sRaw (Fig. 6). (Baseline sRaw values were similar in naive and OVA groups.) Two-factor ANOVA revealed a signi fi cant difference between exposure concentrations (P  0.001), a signi fi cant effect of OVA treatment (P  0.01), and no signi fi cant interaction between factors. Newman-Keuls test revealed that the response was signi fi cantly greater in the OVA-d7

– 10 group than in the naive or OVA-d3 group (which were statistically similar). Interestingly, in the 80-ppm groups, acetic acid did not signi fi cantly increase sRaw over baseline (mean change 0.3  0.2 cmH 2 

### O

 s) in naive animals, whereas in the OVA-d7

– 10 animals Fig. 4. Respiratory rate response to acrolein (minimum breaths/min expressed as percentage of baseline) for naive animals and animals exposed to ovalbumin (OVA) challenge for 3 days (OVA-d3) and 7

– 10 days (OVA-d7

– 10). Breathing frequency averaged

 300 breaths/min during the baseline period. Values are means  SD; data from the OVA-d3 and OVA-d7

– 10 groups are displaced slightly to the left andright, respectively, for clarity. Two-factor ANOVA revealed a significant effect of exposure concentration (P  0.001) and a signi fi cant effect of treatment group (P  0.05); a signi fi cant statistical interaction was not detected. Newman-Keuls test revealed a signi fi cant difference between OVA-d7

– 10 group and control; response of OVA-d3 group was not statistically different from control. Each group consisted of 3

– 6 animals. Fig. 5. Respiratory rate response to acetic acid (minimum breaths/ min expressed as percentage of baseline) for naive, OVA-d3, and OVA-d7

– 10 groups. Breathing frequency averaged

 300 breaths/ min during the baseline period. Values are means  SD; data from OVA-d3 and OVA-d7

– 10 groups are displaced slightly to the left andright, respectively, for clarity. Two-factor ANOVA revealed a significant effect of exposure concentration (P  0.001) and a signi fi cant effect of treatment group (P  0.05); a signi fi cant statistical interaction was not detected. Newman-Keuls test revealed a signi fi cant difference between OVA-d7

– 10 group and control; response of OVA-d3 group was not statistically different from control. Each group consisted of 3

– 8 animals.

Fig. 6. Airway obstructive response to acetic acid [maximum increase in speci fi c airway resistance (sRaw) over baseline] for naı  ̈ve, OVA-d3, and OVA-d7

– 10 groups. Values are means  SD; data from OVA-d3 and OVA-d7

– 10 groups are displaced slightly to the left and right, respectively, for clarity. Baseline sRaw averaged

 2 cmH 2 O

 s. Two-factor ANOVA revealed a signi fi cant effect of exposure concentration (P  0.001) and a signi fi cant effect of treatment group (P  0.05); a signi fi cant statistical interaction was not detected. NewmanKeuls test revealed a signi fi cant difference between OVA-d7

– 10 group and control; response of OVA-d3 group was not statistically different from control. Each group consisted of 3

– 6 animals. 

### 1568 NASAL REFLEX RESPONSES TO ACROLEIN AND ACETIC ACID

J Appl Physiol • VOL 94 • APRIL 2003 • www.jap.orgon September 21, 2009jap.physiology.orgDownloaded from

### this exposure level produced a signi fi cant increase in sRaw (mean change 0.73  0.27 cmH 2 

### O

 s). The designof the experiment was based on use of identical exposure concentrations in the naive and OVA-treated mice. Future experiments would be needed to establish the threshold for acetic acid-induced effects in the OVA-treated animals.

DISCUSSION



### These results elucidate similarities and dissimilarities in the URT responses of the rat and mouse to twoirritants: acrolein and acetic acid. Both vapors produced a sensory irritation response in both species, aresponse known to be directly mediated via nasal trigeminal nerve stimulation (1a, 30). As has been observed for many vapors (9), the RD 50 

### was much lower in the mouse than in the rat for acrolein [1.59 ppm (Fig. 3) vs. 6 ppm (3)] and acetic acid [329 ppm (Fig. 4) vs. 1,040 ppm (39)]. The reasons for the difference are not known. In the rat, the most sensitive URT response to either irritant was vasodilation (28, 29, 39). In this species, the response was quite marked, as indicated by a twofold or greater increase in acetone uptake rates. A small vasodilatory response (20% increase in acetone uptake) was observed in acrolein-exposed mice (Fig. 1) but was not evident in acetic acid-exposed mice. (It was not possible to examine the effect of higher acetic acid exposure concentrations because of URT obstruction.) These results suggest that the mouse iscapable of mounting a vasodilatory response to irritants, but the response is much smaller than in the rat nose. In contrast, a marked URT obstructive response was induced in the mouse by both irritants. A sensory nerve-mediated obstructive response to acrolein (but not acetic acid) occurs in the rat nose (28, 29, 39), but it is delayed in onset and of smaller magnitude than that observed in the mouse. Thus the principal URT response to these irritants appears to be vasodilation in the rat and obstruction in the mouse. The range of sensory nerve responses induced in the human by speci fi c irritant classes (e.g., electrophiles vs. acids) has not been well de fi ned. The breathing frequency and obstructive responses were signi fi cantly attenuated by capsaicin pretreatment, providing strong evidence that sensory nerves were involved in their elicitation. Moreover, because capsaicin acts through the vanilloid receptor (12, 18, 43, 48), these results suggest that the responses were mediated by vanilloid receptor-expressing C fibers. Whether either vapor selectively stimulates IB4  

### or IB4



### nerves is not known. Future studies are needed to determine why sensory nerve stimulation induces a vasodilatory response in the rat vs. an obstructive response in the mouse. The present study included measures in the isolated URT of anesthetized mice and in intact spontaneouslybreathing conscious mice. Similar obstructive responses were observed in both paradigms. Although precise comparisons cannot be made, the magnitude of the obstructive response in the URT was suf fi cient to account for the entire response observed in the intactanimal studies. This suggests that the URT is a significant contributor to the obstructive response observed in intact animals. Because the uptake ef fi ciency of acrolein and acetic acid vapors in the URT exceeded 90%, this is, perhaps, not unsurprising. The URT also appears to be a signi fi cant contributor to the respiratory tract obstructive response to methacholine aerosol (41). Upper airway obstruction may be due to vascularcongestion and/or excessive mucus secretion. For acrolein and acetic acid, the former seems more likely, because the response was immediate and not blocked by atropine and nasal lavage Evans blue dye content was not increased by the irritants (Fig. 3).Irritant-induced breathing frequency and/or obstructive responses were enhanced in animals with OVAinduced allergic airway disease on days 7–10 but not

on day 3 . The mechanism for the enhanced sensory irritation response in allergic animals is not known. It is possible that there is enhanced deposition or uptakeof the inspired irritants in the nasal tissues and increased delivery of irritant to the trigeminal nerve endings in the diseased animals. However, this seems unlikely, because URT uptake ef fi ciencies for these
vapors were  90% in the healthy animals. The time course for the irritant sensitivity appears to correlate with the airway in fl ammation, inasmuch as the eosinophilic infi ltration is not fully developed in this model until days 7–10 (21, 34, 50). In this model, airway hyperresponsiveness to methacholine is demonstrable at day 3 , as measured by pulmonary resistance, gas trapping, or enhanced pause (34, 49). Moreover, ourrecent studies have shown an enhanced URT obstructive response to methacholine on day 3 (41). In contrast, irritant sensitivity is not apparent until days 7–10 in this model, suggesting that irritant sensitivity and methacholine hyperresponsiveness are mediated via different mechanisms.The precise nature of the alterations in the sensitivity to the irritants that was observed in the OVAtreated animals was irritant speci fi c. Speci fi cally, although the breathing pattern response was enhancedfor both irritants, the obstructive response was enhanced for only acetic acid, not acrolein. If a single mechanism were responsible, it would be anticipatedthat breathing frequency and airway obstructive responses would demonstrate similar modulations for both vapors in the OVA-treated animals. That this wasnot the case suggests involvement of multiple pathways or mechanisms. Because alteration in breathing pattern is a neurally mediated afferent re fl ex response (1a, 30), it is likely that the enhanced responsiveness

re fl ects an alteration in neural sensitivity and/or neural integration in the central nervous system (45),rather than an increased local release of neuropeptides. It is likely that acidic irritants stimulate sensory nerves through the vanilloid receptor (12, 13), and thecapsaicin pretreatment studies indicate a role for vanilloid receptor-expressing nerves in the response. [The responses to acetic acid were not likely to be due to the acetate, inasmuch as the response to sodium 1569 NASAL REFLEX RESPONSES TO ACROLEIN AND ACETIC ACID

J Appl Physiol • VOL 94 • APRIL 2003 • www.jap.orgon September 21, 2009jap.physiology.orgDownloaded from

### acetate aerosol (1,000  g/l) was quite mild and nogreater than the response to equimolar hypertonic saline aerosol (data not shown).] Acrolein may activate the vanilloid receptor as well (43). Perhaps there is analteration in vanilloid receptor expression and/or function in this model of allergic airway disease. Re fl ex(sneeze) and local (plasma protein extravasation) responses to the vanilloid receptor agonist capsaicin are enhanced in human subjects with rhinitis (33). Nerve growth factor (NGF) expression is enhanced in allergic airway infl ammation (10, 33), and NGF has been shown to infl uence vanilloid receptor expression (19, 23, 47).

The airway obstructive response to acetic acid, but not acrolein, vapor was enhanced in the OVA-treated animals. Because the sensory irritation response toboth vapors is enhanced, it is not likely that heightened obstructive response to acetic acid is not simplydue to enhanced neural sensitivity and/or neural integration. It has recently been shown that the airwayobstructive response to the irritant capsaicin is enhanced in a guinea pig allergic airway disease model (8) and that the nasal mucus secretory response to capsaicin is enhanced in human subjects with rhinitis (33). Thus the effects observed in the present study do not appear to be speci fi c to the mouse. Sensory nervedependent obstruction in rodents may be mediated by axonal neuropeptide release (4, 5, 22, 37). Recent data suggest an increased expression of neuropeptides in airway sensory nerves in allergic infl ammation, perhaps due to enhanced NGF release (17, 19, 33). This

may re fl ect elevated expression in A

- fibers (19). However, enhanced neuropeptide-mediated responses mayalso result from a variety of extraneuronal mechanisms, including alterations in neuropeptide receptorexpression and/or inhibition of neuropeptide catabolism via peptidases (1, 5, 32, 45). Perhaps the enhancedbreathing frequency response is due to increased vanilloid receptor expression and the enhanced obstructive response is due to nerve type-speci fi c (A - vs. C fiber or IB4  

### vs. IB4



### ) increases in neuropeptide ex

pression. Future studies are needed to clarify these possibilities.Irrespective of the precise mechanisms, the responses to acetic acid in the mouse model appear to be similar to those observed in the human. Speci fi cally, patients with asthma have a reduced threshold for sulfuric acid-induced airway obstruction (46), and mice with OVA-induced allergic disease respond to 80 ppm acetic acid, a concentration that is without effect in the control mice. It is not known whether the responses to high concentrations of irritants in animal models are

re fl ective of those of humans exposed to lower concentrations; however, this murine model may provide a useful tool for investigating potential mechanisms ofinduction of irritant sensitivity by allergic airway disease.

The expert technical assistance of B. Simmons is gratefully acknowledged. This work was supported by National Institute of Environmental Health Sciences Grant RO1 ES-08765. Its contents are solely the responsibility of the authors and do not necessarily represent the of

fi cial views of the National Institute of Environmental Health Sciences.

REFERENCES

1. Adcock IM, Peters M, Gelder C, Shirasaki H, Brown CR, and Barnes PJ. Increased tachykinin receptor gene expression in asthmatic lung and its modulation by steroids. Mol Endocrinol 11: 1

– 7, 1993. 1a. Alarie Y. Sensory irritation by airborne chemicals . CRC Crit

Rev Toxicol 3: 299

– 363, 1973. 2. Alarie Y. Bioassay for evaluating the potency of airborne sensory irritants and predicting acceptable levels of exposure in

man. Food Cosmet Toxicol 19: 623

– 626, 1981. 3. Babiuk C, Steinhagen WH, and Barrow CS. Sensory irritation response to inhaled aldehydes after formaldehyde pretreatment. Toxicol Appl Pharmacol 79: 143

– 149, 1985. 4. Barnes PJ. Role of neural mechanisms in airway defense. In: Environmental Impact on the Airways, edited by Cretien J and Dusser D. New York: Dekker, 1996, p. 93

– 121. 5. Barnes PJ. Neurogenic infl ammation in the airways. Respir Physiol 125: 145

– 154, 2001. 6. Barnes PJ, Baraniuk JN, and Belvisi MG. Neuropeptides in the respiratory tract

. Part I . Am Rev Respir Dis 144: 1187

– 1198, 1991. 7. Barnes PJ, Baraniuk JN, and Belvisi MG. Neuropeptides in the respiratory tract. Part II . Am Rev Respir Dis 144: 1391

– 1399, 1991. 8. Bergren DR. Chronic tobacco smoke exposure increases airway sensitivity to capsaicin in awake guinea pigs. J Appl Physiol 90: 695

– 704, 2001. 9. Bos PM, Zwart A, Reuzel PB, and Bragt PC. Evaluation of the sensory irritation test for the assessment of occupational health risk. Crit Rev Toxicol 21: 423

– 450, 1996. 10. Braun A, Lommatzsch M, Lewin GR, Virchow JC, and Renz H. Neurotropins: a link between airway infl ammation and airway smooth muscle contractility in asthma? Int Arch Allergy Immunol 118: 163

– 165, 1999. 11. Cander L and Forster RE. Determination of pulmonary parenchymal tissue volume and pulmonary capillary blood fl ow in

man. J Appl Physiol 14: 541

– 551, 1959. 12. Caterina MJ and Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Annu Rev Neurosci 24: 487

– 517, 2001. 13. Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M, Basbaum AI, and Julius D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288: 306

– 312, 2000. 14. Chung K, Klein CM, and Coggeshall RE. The receptive part of the primary afferent axon is most vulnerable to systemic capsaicin in adult rats. Brain Res 511: 222

– 226, 1990. 15. Coleridge JC and Coleridge HM. Afferent vagal C fibre innervation of the lungs and airways and its functional signi fi

cance. Rev Physiol Biochem Pharmacol 99: 1

– 110, 1984. 16. Fish JE and Peters SP. Asthma: clinical presentation and management. In: Fishman ’ s Pulmonary Diseases and Disorders (3rd ed.), edited by Fishman AP, Elias JA, Fishman JA, Grippi MA, Kaiser LR, and Senior RM. New York: McGraw-Hill, 1998, p. 757

– 776. 17. Fisher A, McGregor G, Saria A, Philippin B, and Kummer W. Induction of tachykinin gene and peptide expression in guinea pig nodose primary afferent neurons by allergic in fl ammation. J Clin Invest 98: 2284

– 2291, 1996. 18. Holzer P. Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol Rev 43: 143

– 201, 1991. 19. Hunter DD, Myers AC, and Undem BJ. Nerve growth factorinduced phenotypic switch in guinea pig airway sensory neurons. Am J Respir Crit Care Med 161: 1985

– 1990, 2000. 20. Hunter DD and Undem BJ. Identi fi cation and substance P content of vagal afferent neurons innervating the epithelium of 

### 1570 NASAL REFLEX RESPONSES TO ACROLEIN AND ACETIC ACID

J Appl Physiol • VOL 94 • APRIL 2003 • www.jap.orgon September 21, 2009jap.physiology.orgDownloaded fromthe guinea pig trachea. Am J Respir Crit Care Med 159: 1943

– 1948, 1999. 21. Jacobson SB, Symanowicz PT, Hubbard AK, Whiteley HE, Thrall RS, Cloutier MM, and Morris JB. Effect of nasalirritants on ovalbumin-induced allergic airway disease in a murine model (Abstract). Toxicol Sci 60S: 71, 2001. 22. Lundberg JM. Tachykinins, sensory nerves, and asthma

— an overview. Can J Physiol Pharmacol 73: 908

– 914, 1995. 23. Michael GJ and Priestley JV. Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy. J Neurosci 19: 1844

– 1854, 1999. 24. Morris JB. Dosimetry

, toxicity and carcinogenicity of inspired acetaldehyde in the rat. Mutat Res 380: 113

– 124, 1997. 25. Morris JB. A method for measuring upper respiratory tract vapor uptake and its applicability to quantitative inhalation risk assessment. Inhal Toxicol 11: 943

– 965, 1999. 26. Morris JB. Sensory nerve-mediated nasal vasodilatory response to inspired ethyl acrylate. Inhal Toxicol 14: 585

– 597, 2001. 27. Morris JB, Gianutsos G, and Stanek J. Sensory-nerve mediated nasal vasodilatory response to ethyl acrylate (Abstract). Toxicol Sci 54S: 312, 2000. 28. Morris JB, Stanek J, and Gianutsos G. Sensory nerve-mediated immediate nasal responses to inspired acrolein. J Appl Physiol 87: 1877

– 1886, 1999. 29. Morris JB, Stanek J, and Gianautsos G. Acrolein-induced nasal vasodilation is CGRP- and NO-mediated (Abstract). Toxicol Sci 48S: 173, 1999. 30. Nielsen GD. Mechanisms of activation of the sensory irritant receptor by airborne chemicals. Crit Rev Toxicol 21: 183

– 208, 1991. 31. Petersson G, Bacci E, McDonald DM, and Nadel JA. Neurogenic plasma extravasation in the rat nasal mucosa is potentiated by peptidase inhibitors. J Pharmacol Exp Ther 264: 509

– 514, 1993. 32. Piedimonte G. Tachykinin peptides, receptors and peptidases in airway disease. Exp Lung Res 21: 809

– 834, 1995. 33. Sanico AM, Koliatsos VE, Stanisz AM, Bienenstock J, and Togias A. Neural hyperresponsiveness and nerve growth factor in allergic rhinitis. Int Arch Allergy Immunol 118: 154

– 158, 1999. 34. Schramm CM, Puddington L, Yiamouyiannis CA, Lingenheld EG, Whiteley HE, Wolyniec WW, Noonan TC, and Thrall RS. Proinfl ammatory roles of T-cell receptor (TCR)  and TCR lymphocytes in a murine model of asthma. Am J Respir Cell Mol Biol 22: 218

– 225, 2000. 35. Scuri M, McCaskill V, Chediak AD, Abraham WM, and Wanner A. Measurement of airway mucosal blood fl ow with dimethyl ether: validation with microspheres. J Appl Physiol 79: 1386

– 1390, 1995. 36. Silver WL, Farley LG, and Finger TE. The effects of neonatal capsaicin administration on trigeminal nerve chemoreceptors in the rat nasal cavity. Brain Res 561: 212

– 216, 1991. 37. Spina D, Shah S, and Harrison S. Modulation of sensory nerve function in the airways. Trends Pharmacol Sci 19: 460

– 466, 1998. 38. Stanek J and Morris JB. The effect of inhibition of aldehyde dehydrogenase on nasal uptake of inspired acetaldehyde. Toxicol Sci 49: 225

– 231, 1999. 39. Stanek J, Symanowicz PT, Olsen JE, Gianutsos G, and Morris JB. Sensory-nerve-mediated nasal vasodilatory response to inspired acetaldehyde and acetic acid vapors. Inhal Toxicol 13: 807

– 822, 2001. 40. Stucky CL and Lewin GR. Isolectin B 4 -positive and -negative nociceptors are functionally distinct. J Neurosci 19: 6497

– 6505, 1999. 41. Symanowicz PT, Hubbard AK, and Morris JB. Methacholine aerosol-induced nasal obstruction in the mouse (Abstract). Toxicol Sci 66S: 479, 2002. 42. Szallasi A and Blumberg PM. [ 3 H]resiniferatoxin binding bythe vanilloid receptor: species-related differences: effects of temperature and sulfhydryl reagents. Naunyn Schmiedebergs Arch Pharmacol 347: 84

– 91, 1993. 43. Szallasi A and Blumberg PM. Vanilloid (capsaicin) receptors and mechanisms. Pharmacol Rev 51: 159

– 212, 1999. 44. Undem BJ and Carr MJ. Pharmacology of airway afferent nerve activity. Respir Res 2: 234

– 244, 2001. 45. Undem BJ, Kajekar R, Hunter DD, and Meyers C. Neural integration and allergic disease. J Allergy Clin Immunol 106: S213

– S220, 2000. 46. Utell MJ, Morrow PE, Speers DM, Darling J, and Hyde RW. Airway responses to sulfate and sulfuric acid aerosols in asthmatics: an exposure-response relationship. Am Rev Respir Dis 128: 444

– 450, 1983. 47. Winston J, Toma H, Shenoy M, and Pasricha PJ. Nerve growth factor regulates VR-1 mRNA levels in cultures of adult dorsal root ganglion neurons. Pain 89: 181

– 186, 2001. 48. Wood JN and Docherty R. Chemical activators of sensory

neurons. Annu Rev Physiol 59: 457

– 482, 1997. 49. Wu CA, Puddington L, Whiteley HE, Yiamouyiannis CA, Schramm CM, Mohammadu F, and Thrall RS. Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease. J Immunol 167: 2798

– 2807, 2001. 50. Yiamouyiannis CA, Schramm CM, Puddington L, Stengel P, Baradaran-Hosseini E, Wolyniec WW, Whiteley HE, and Thrall RS. Shifts in lung lymphocyte pro fi les correlate with the sequential development of acute allergic and chronic tolerant stages in a murine asthma model. Am J Pathol 154: 11

– 21, 1999. 

### 1571 NASAL REFLEX RESPONSES TO ACROLEIN AND ACETIC ACID

J Appl Physiol • VOL 94 • APRIL 2003 • www.jap.orgon September 21, 2009jap.physiology.orgDownloaded from