Study design: We performed a randomized, two-way crossover study in subjects with seasonal allergic rhinitis out of season. Subjects came to the Nasal Physiology Laboratory for screening, where they completed an allergy questionnaire and underwent skin-prick testing for confirmation of a grass or ragweed allergy. The skin test included positive and negative controls and the results were graded compared with the controls as 1+ to 4+ (1+: wheal larger than negative control and smaller than positive control; 2+: wheal 5-7 mm; 3+: wheal 7-10 mm; 4+: any reaction with a wheal \>10 mm or pronounced pseudopodia). Subjects with positive skin test (between 2+ and 4+) and a positive history of allergic symptoms during the relevant seasons then underwent a screening nasal challenge with either grass or ragweed allergen. Subjects who passed the screening challenge (twofold increase in either ipsilateral or contralateral nasal secretions after allergen challenge compared with diluent) had a 2-week washout period and returned to the laboratory, where they were randomized to receive intranasal treatment with either CO2 or no treatment. Thirty minutes after treatment, subjects underwent a nasal challenge with allergen. Seven subjects were challenged with ragweed and five subjects were challenged with grass. Subjects had another 2-weeks washout period and were then crossed over to the other treatment followed by a similar challenge. Previous work in our laboratory using a similar challenge system showed that allergen-induced inflammatory changes are back to baseline 2 weeks after the challenge.

Subjects: Twelve subjects participated. Subjects were studied outside their allergy season. All subjects were healthy except for mild asthma requiring only as-needed bronchodilators. They were not on any medications and had not received antihistamines or leukotriene receptor antagonists for at least 1 week and intranasal steroids for at least 1 month before enrollment and for the duration of the study.

Treatment: CO2 was applied for 10 seconds in each nostril using a special applicator (plastic tight-seal nosepiece) attached to a CO2 canister and a flow control valve. It was delivered at a flow rate of 0.5 standard L/min with the mouth open to prevent inhalation. Thus, for 20-second duration of administration, the total dose of CO2 delivered was 167 mL. The amount of CO2 delivered to the mucosa is unknown. The no-treatment arm involved placement of the device but no gas was delivered. Therefore, neither the subjects nor the investigators were blinded to the treatment administered. The no-treatment arm did not involve the delivery of air without CO2 to the nose because we were concerned that blowing dry air into the nose might cause a mucosal reaction that could confound the results. We have previously shown that cold, dry air challenges create a hyperosmolar environment, triggers mast cell activation, and induces a nasonasal reaction.7 Thus, because our primary outcome was the objective measure of the nasonasal reflex, we avoided this possibility.

Nasal Challenge: The subjects were allowed 15 minutes to acclimatize to the laboratory environment before challenge. Baseline sneezes reflecting the 15 minutes of acclimatization and nasal and eye symptoms were recorded followed by collection of a nasal scraping for quantitation of eosinophils in nasal secretions. Sneezes were recorded by the subjects during each of the assessed intervals of the challenge protocol. The subjects were reminded to keep track of the number of sneezes by the research coordinator who was present for the duration of the challenges. Intranasal CO2 or sham was then applied for 10 seconds to each nostril. Thirty minutes later, sneezes and symptoms were recorded again, to reflect the 30-minute time period, and nasal challenge was initiated. Because we were interested in evaluating the effect of the treatment on allergen-induced nasal reflexes, we used filter paper disks to perform the challenges and monitor the secretory response as previously described.

Briefly, 8-mm filter paper disks (Shandon, Inc., Pittsburgh, PA) were used for both nasal challenge and collection of resultant secretions. They were placed on the anterior nasal septum, beyond the mucocutaneous junction, under direct vision using a nasal speculum, forceps, and a headlight. Fifty microliters of challenge solutions were placed on the disks, which were then applied to the nasal septum for 1 minute. Thirty seconds after removal, two preweighed filter paper disks were placed on both sides of the nasal septum for 30 seconds, collecting nasal secretions from the challenge site (ipsilateral) and the contralateral nostril. These disks were then immediately placed back into microtubes and weighed. The difference in their weight before and after challenge was the weight of produced nasal secretions, which was recorded in milligrams. Ten minutes after each challenge, the number of sneezes as well as symptoms on each side were recorded by the subjects reflective of the 10-minute interval The first challenge was performed using phenol-buffered saline, the diluent for the allergen extracts, and this was followed by 2 increasing doses of grass or ragweed allergen. The time from treatment administration to the first allergen challenge was approximately 40 minutes and to the second allergen challenge, 50 minutes. The amount of allergen applied on the paper disks for challenge were 333 and 1000 BAU (bioequivalent allergy unit) of grass allergen extract (Hollister-Stier, Spokane, WA) or 50 microliters of ragweed antigen extract at concentrations of 1:666 and 1:200 w/v (Hollister-Stier). The subjects came back to the laboratory 24 hours later and underwent a scraping of their nasal secretions to evaluate for eosinophil influx.