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

M. Cloutier and Andrea K. Hubbard 

John B. Morris, Peter T. Symanowicz, Joshua E. Olsen, Roger S. Thrall, Michelle

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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. Hubbard1

1

University of Connecticut Pulmonary Research Consortium, Department of Pharmaceutical

Sciences, University of Connecticut, Storrs 06269; and Departments of 2

Medicine and 3

Pediatrics, 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) vapors

induced 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 these

responses. 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 mechanosensory

function, whereas C fibers are important in the detection of irritant chemicals (15, 30, 44). The distinction is

not 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:

IB4and 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 reflex

responses 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 (RD50) (1a). Capsaicin is a potent sensory irritant (1a, 30); in our hands a 50% reduction in

breathing 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 are

mediated, in part, by release of neuropeptides via antidromal stimulation (4–7, 32). Presumably the neuropeptide-rich IB4fibers (40) are most important in

these 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.

http://www.jap.org 8750-7587/03 $5.00 Copyright © 2003 the American Physiological Society 1563

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nerve-mediated responses to inspired irritants in the

rat and included measures of afferent reflex responses

(changes in breathing pattern) and local tissue responses, including neurogenic edema, airway obstruction, and mucus secretion (26, 28, 39). (The URT is

defined as all portions of the respiratory tract anterior

to 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 acidification is believed to be important, acetic

acid vapor and acetaldehyde. [Acetaldehyde is metabolized in nasal tissues to acetic acid via aldehyde dehydrogenase, and this is believed to be a critical step in

its 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, confirming 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) to

compare 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 endotracheal

tube. 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 flow

rate (25 ml/min) for 50 min. Chamber air also contained acetone vapor. Uptake of acetone vapor was

monitored 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 flow 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 fluid (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 efficiency 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 flow resistance, rather than

vasodilation, as observed in the rat. Because airway

obstruction 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 was

examined 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. Some

studies 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°C

with 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 Scientific, Springfield, 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 to

allow for equilibration. Chamber concentrations were controlled by changing the irritant concentrations in the aqueous generation fluids. 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 or

plethysmograph 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

flame ionization detection. A 15-m DB-WAX column (J & W

Scientific, Folsom, CA) was used with a column temperature

of 33°C and a carrier gas (N2) flow 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 flow 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 first, indicating the high collection efficiency of the

system.) Analysis was performed using a mobile phase

consisting of 5:95 vol/vol acetonitrile-0.1% H3PO4 at a flow

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), flow 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 RD50

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based on pilot studies. Our laboratory’s previous studies on

the rat indicated that local tissue responses were easily

demonstrable at RD50 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 animal

was then placed in the nose-only chamber in a supine position, and chamber air was drawn through the isolated URT

at a constant flow rate of 25 ml/min for 50 min, which was the

highest flow 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 to

prevent 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 fluid 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 fluorometrically 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). Briefly, vapor

concentrations in chamber air (Cin) and in air that had been

drawn through the isolated URT (Cex) were measured by gas

chromatography as described above. Cin was measured immediately 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 into

the gas chromatograph at 3-min intervals to provide continuous monitoring of Cex during exposure. Acetone uptake

rates were calculated from the ratio of Cex to Cin and normalized to an inspired concentration of 44 ppm (100 g/l).

URT flow resistance was also monitored throughout exposure. Toward this end, the endotracheal tube contained a “T,”

one side of which was connected to a differential pressure

transducer (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

flow rate (25 ml/min).

In a separate group of mice, URT uptake efficiencies 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 (flow rate 25 ml/min), and irritant concentration was

measured in the URT exiting air (Cex). Acrolein concentration 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 fluid was analyzed for

acetic acid content by HPLC, as described above. The uptake

efficiency was calculated from the inspired (chamber) air

concentration (Cin) and the URT exiting air concentration

(Cex) 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 and

300 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

first anesthetized with tribromoethanol (Avertin) and then

treated 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 double

plethysmograph (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 exposure

and through the head-space side of the double plethysmograph at a flow rate of 0.4 l/min. Three responses were

measured: breathing rate, expiratory pause duration, and

specific 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

(cmH2O 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). Briefly, 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-airflow nose-only

inhalation chamber (CH Technologies). Atmospheres were

generated by nebulization (Lovelace Nebulizer, In-Tox Products, Albuquerque, NM) of 1% OVA in saline. Aerosol concentration averaged 20 mg/m3 (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 breathing

parameters 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 time

points in the OVA model were selected. Animals were examined 24 h after the third daily OVA aerosol challenge expoNASAL REFLEX RESPONSES TO ACROLEIN AND ACETIC ACID 1565

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sure (OVA-d3) to examine a time when the airway inflammation 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

inflammation 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 were

compared among groups by one- or two-factor ANOVA followed by Newman-Keuls test. Animal groups consisted of

three to eight animals. Repeated-measures ANOVA was performed on the URT uptake and resistance data. RD50 values

were calculated by logarithmic linear regression of breathing

frequency vs. exposure concentration data (1a). P  0.05 was

required for significance. 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 acetic

acid, respectively. The effects of the irritants on acetone vapor uptake are shown in Fig. 1. Repeatedmeasures ANOVA revealed that acrolein produced a

significant 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 increase

may have occurred, acetic acid was without statistically significant effect on uptake. URT flow resistance

during exposure to these irritants is shown in Fig. 2.

Both vapors produced a rapid and statistically significant increase in URT flow 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 cmH2Oml1  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 significant

plasma protein extravasation and/or mucus secretion

during exposure.

The URT uptake efficiency studies revealed that

acrolein and acetic acid were scrubbed from the airstream with high efficiency in the URT. Acrolein exposure concentration (Cin) 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 left

and right, respectively, for clarity. Repeated-measures ANOVA revealed no statistical effect of exposure time, a significant 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

significantly elevated over control levels. Each group consisted of

9–12 animals.

Fig. 2. URT flow resistance (mean  SD) during exposure to clean

air (control), 1.1 ppm acrolein, or 330 ppm acetic acid. Acrolein and

acetic acid data are displaced slightly to the left and right, respectively, for clarity. Repeated-measures ANOVA revealed no statistical

effect of exposure time, a significant difference between exposure

groups (P  0.05), and no statistical interaction between exposure

group and time. Newman-Keuls test revealed that flow resistance in

the acrolein and acetic acid groups was significantly 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 significant differences among exposure groups. Each

group consisted of 9–12 animals.

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air. On the basis of the limit of detection of the gas

chromatographic technique of 0.1 ppm, URT uptake

efficiencies 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 efficiency 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 cmH2O s, respectively. Both vapors caused

a significant decrease in respiratory rate, a significant

expiratory pause, and a significant increase in sRaw

(Table 1). The increases in respiratory resistance in

absolute terms (cmH2Oml1  s) are not known, inasmuch 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

cmH2Oml1  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 flow 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 significant difference between vapors, with

acetic acid producing larger responses, and a significant effect of capsaicin pretreatment, with significantly

lower responses in the 20  50 and 20  75 groups

than in the vehicle control or 25  25 group (NewmanKeuls test). A significant 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 significant 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 cmH2O s, respectively, in vehicletreated mice compared with 55  7% of baseline, 244 

47 ms, and 0.92  0.78 cmH2O s, respectively, in

atropine-pretreated mice. For acrolein, these values

averaged 58  18% of baseline, 215  124 ms, and

1.48  0.82 cmH2O s, respectively, in vehicle-treated

mice compared with 49  4% of baseline, 268  52 ms,

and 1.00  0.86 cmH2O s, respectively, in atropinepretreated mice.

Concentration response in healthy and allergic airway-diseased mice. The effect of acrolein exposure on

breathing 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 significant 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-d3

animals were statistically similar and that the response was greater in the OVA-d7–10 than in the

na ̈ıve or OVA-d3 animals. RD50 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 554 6313 7715*† 8511*†

Acetic acid 476 539 7114*† 766*†

Expiratory pause, ms

Acrolein 20266 15571* 6494*† 119*†

Acetic acid 32139 25038* 15933*† 11927*†

sRaw, cmH2Os

Acrolein 0.560.34 0.580.48 0.130.23* 0.090.15*†

Acetic acid 2.060.70 1.470.55 1.080.29* 0.890.15*†

Values are means  SD of 4–6 mice. Expiratory pause and specific 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 cmH2Os, respectively. In

vehicle-treated animals, respiratory rates were significantly 100% of baseline, and increases in expiratory pause duration and sRaw were

significantly  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 significant difference between irritants was detected for each measure (P  0.002 in all cases). A significant 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).

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0.40 cmH2O 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

cmH2O 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 significant difference between exposure concentrations (P  0.001), a significant difference between OVA groups (P  0.01), and no significant 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. RD50 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 significant difference between exposure concentrations (P  0.001), a significant effect of OVA

treatment (P  0.01), and no significant interaction

between factors. Newman-Keuls test revealed that the

response was significantly 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 significantly increase sRaw

over baseline (mean change 0.3  0.2 cmH2O 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 and

right, respectively, for clarity. Two-factor ANOVA revealed a significant effect of exposure concentration (P  0.001) and a significant

effect of treatment group (P  0.05); a significant statistical interaction was not detected. Newman-Keuls test revealed a significant

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 and

right, respectively, for clarity. Two-factor ANOVA revealed a significant effect of exposure concentration (P  0.001) and a significant

effect of treatment group (P  0.05); a significant statistical interaction was not detected. Newman-Keuls test revealed a significant

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 specific airway resistance (sRaw) over baseline] for na ̈ıve,

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 cmH2O s.

Two-factor ANOVA revealed a significant effect of exposure concentration (P  0.001) and a significant effect of treatment group (P 

0.05); a significant statistical interaction was not detected. NewmanKeuls test revealed a significant 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.

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this exposure level produced a significant increase in

sRaw (mean change 0.73  0.27 cmH2O s). The design

of 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 two

irritants: acrolein and acetic acid. Both vapors produced a sensory irritation response in both species, a

response known to be directly mediated via nasal trigeminal nerve stimulation (1a, 30). As has been observed for many vapors (9), the RD50 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 is

capable 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

specific irritant classes (e.g., electrophiles vs. acids)

has not been well defined.

The breathing frequency and obstructive responses

were significantly 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

IB4nerves 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 spontaneously

breathing 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 sufficient to

account for the entire response observed in the intact

animal studies. This suggests that the URT is a significant contributor to the obstructive response observed

in intact animals. Because the uptake efficiency of

acrolein and acetic acid vapors in the URT exceeded

90%, this is, perhaps, not unsurprising. The URT also

appears to be a significant contributor to the respiratory tract obstructive response to methacholine aerosol

(41). Upper airway obstruction may be due to vascular

congestion 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 uptake

of 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 efficiencies for these

vapors were 90% in the healthy animals. The time

course for the irritant sensitivity appears to correlate

with the airway inflammation, inasmuch as the eosinophilic infiltration 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, our

recent 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 specific. Specifically, although the breathing pattern response was enhanced

for both irritants, the obstructive response was enhanced for only acetic acid, not acrolein. If a single

mechanism were responsible, it would be anticipated

that breathing frequency and airway obstructive responses would demonstrate similar modulations for

both vapors in the OVA-treated animals. That this was

not the case suggests involvement of multiple pathways or mechanisms. Because alteration in breathing

pattern is a neurally mediated afferent reflex response

(1a, 30), it is likely that the enhanced responsiveness

reflects 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 the

capsaicin 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

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acetate aerosol (1,000 g/l) was quite mild and no

greater than the response to equimolar hypertonic saline aerosol (data not shown).] Acrolein may activate

the vanilloid receptor as well (43). Perhaps there is an

alteration in vanilloid receptor expression and/or function in this model of allergic airway disease. Reflex

(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 inflammation (10, 33), and NGF has been

shown to influence 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 to

both vapors is enhanced, it is not likely that heightened obstructive response to acetic acid is not simply

due to enhanced neural sensitivity and/or neural integration. It has recently been shown that the airway

obstructive 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 specific 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 inflammation, perhaps due to enhanced NGF release (17, 19, 33). This

may reflect elevated expression in A-fibers (19). However, enhanced neuropeptide-mediated responses may

also result from a variety of extraneuronal mechanisms, including alterations in neuropeptide receptor

expression and/or inhibition of neuropeptide catabolism via peptidases (1, 5, 32, 45). Perhaps the enhanced

breathing frequency response is due to increased vanilloid receptor expression and the enhanced obstructive response is due to nerve type-specific (A- vs. C

fiber or IB4 vs. IB4) increases in neuropeptide expression. 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. Specifically,

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

reflective of those of humans exposed to lower concentrations; however, this murine model may provide a

useful tool for investigating potential mechanisms of

induction 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

official views of the National Institute of Environmental Health

Sciences.

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