Abstract
Background
There have been few studies about the effect of histamine on ciliary beat frequency (CBF) in the acute phase of allergic rhinitis.
Objective
The present study was designed to investigate CBF in the acute phase of allergic rhinitis and the effect of histamine on CBF.
Materials and Methods
Nasal septal mucosae were obtained from 13 mice that had been systemically immunized and locally challenged with ovalbumin (OVA) (group A), 11 OVA immunized and phosphate-buffered saline–challenged mice (group B), and 12 nontreated negative control mice (group C). The CBFs were observed within 20 minutes of local challenge with OVA. Ciliary beat frequencies were measured before and after treatment with 10 −5 , 10 −3 , and 10 −1 mol/L histamine and after administering antihistamine or Dulbecco’s modified Eagle’s medium to histamine-exposed mucosa.
Results
The baseline CBF in group A was higher than in groups B and C. After treatment with 10 −1 mol/L histamine, CBF in all groups decreased to 0 within 5 to 7 minutes, whereas treatment with 10 −5 or 10 −3 mol/L histamine had no effect. The application of 100 μ mol/L fexofenadine or Dulbecco’s modified Eagle’s medium solution restored histamine-induced ciliostasis to near baseline CBF after 10 minutes.
Conclusions
Ciliary beat frequency increased in the acute phase of a mouse model of allergic rhinitis. Physiologic concentrations of histamine had no effect on CBF, and thus, it appears that other mechanisms control CBF in the acute phase of allergic rhinitis.
1
Introduction
Mucociliary clearance is a critical aspect of host airway defense, and effective clearance requires appropriate mucus production and coordinated ciliary activity. Furthermore, mucociliary clearance is altered by invading virulent organisms, such as viruses and bacteria, and by temperature changes, drugs, and various inflammatory mediators . Decreases in the nasal ciliary beat frequency (CBF) attenuate response to external stimuli and reduce secretion in the nasal cavity and sinus. In addition, such decreases can cause upper airway diseases, such as rhinosinusitis, by obstructing the natural ostium. There are many studies of the relationship between sinusitis and CBF . However, only a few studies have been conducted on CBF in allergic rhinitis, and those performed have produced disparate results. In one study, CBF in allergic rhinitis was found to decline with duration of morbidity, especially in cases of perennial allergic rhinitis . In another study, an increase in CBF was observed when sensitized sheep trachea was challenged with antigen in vivo and in vitro, and this phenomenon was presumed to be caused by chemical mediators secreted from mast cells .
Some experiments have been performed to identify the mediators that could decrease CBF and subsequently inhibit mucociliary clearance . In particular, histamine is known to play a major role in many processes that occur during the acute phase of allergic rhinitis. However, there has been no consensus regarding the effect of histamine on CBF. Some studies have reported stimulatory effects of histamine on CBF, whereas other studies failed to find an effect . Moreover, it is difficult to compare these previous results because of the different materials and methods used. Therefore, we investigated CBF in the acute phase of allergic rhinitis and the effect of histamine on CBF.
2
Methods
2.1
Sensitization of mice and preparation of septal mucosa
2.1.1
Development of mouse model of allergic rhinitis
Thirty-six 4-week-old specific pathogen-free female BALB/c mice weighing 10 to 13 g were used in this study. All mice were treated intraperitoneally (IP) and then challenged intranasally with ovalbumin (OVA) or phosphate-buffered saline (PBS) according to the protocol depicted in Fig. 1. Briefly, group A (n = 13) mice were systemically sensitized with 25 μ g OVA IP dissolved in 300 μ L of PBS containing 2 mg alum (Pierce Biotechnology, Inc, Rockford, IL) as an adjuvant on experimental days 0, 7, and 14. One week after the third IP sensitization, mice were challenged intranasally with OVA for 7 consecutive days. Group B (n = 11) mice were systemically immunized in the same way as group A mice and were challenged intranasally with 20 μ L of PBS for 7 consecutive days. Group C (n = 12) mice were administered PBS IP and intranasally as depicted in Fig. 1.
2.1.2
Observed nasal rubbing frequencies during the first 5 minutes after allergen challenge
Twenty-four hours after the final nasal challenge, 200 μ g OVA was instilled into nasal cavities to provoke allergic symptoms and the numbers of nasal rubbings during the 5 minutes immediately after provocation were counted.
2.1.3
Preparation of nasal septal mucosa
Mice were killed, and both nasal septal mucosae (size range, 2–3 × 3–4 mm) were harvested. Harvested nasal septal mucosae were washed in Dulbecco’s modified Eagle’s medium (DMEM) solution (Dulbecco’s modified Eagle’s medium—Ham’s nutrient F12; Gibco BRL, Grand Island, NY) to remove secretions and blood then placed in the same medium during experiment. Penicillin 10 IU/mL, streptomycin 10 IU/mL, and amphotericin B 0.025 mg/mL were added to the DMEM solution, which was kept at 37°C throughout the procedure.
2.2
Measurement of CBF
We measured CBFs using a digitalized computer analysis system . Harvested septal mucosae were fixed at the center of the base of 35 mm plastic culture dishes using a white gold ring and dental floss. Ciliary beat frequencies were measured using an immersion microscope at ×400 in areas in which cilia were found to be moving actively. A digital charge-coupled camera (XC-HR50; Sony Co, Tokyo, Japan) capable of operating at 60 full frames per second transferred images to a computer equipped with a frame grabber (PCVisionplus; Dalsa Co, St. Laurent, Canada). Ciliary movement was analyzed using software based on fast Fourier transform.
2.3
Design of experiment
2.3.1
Measurement of CBF in vitro within 20 minutes after in vivo nasal allergen challenge
Mucosae were harvested from the septum after euthanizing to measure CBF in vitro, within 20 minutes after in vivo nasal allergen challenge. Ciliary beat frequencies were measured at 0, 5, 10, and 15 minutes.
2.3.2
The effect of histamine on CBF
After measuring CBF for 15 minutes, solutions containing 10 −5 , 10 −3 , or 10 −1 mol/L histamine in DMEM were perfused into cultures to replace the preexisting DMEM solution. Ciliary beat frequency was measured at 0, 5, 10, and 15 minutes after histamine treatment.
2.3.3
Ciliary beat frequency responses after treatment with fexofenadine and DMEM solution
To assess the effect of histamine, CBF was measured after treating histamine-exposed tissue with antihistamine or DMEM solution. Fexofenadine (HanDok Co, Seoul, South Korea) was used as the antihistamine, and a concentration of 100 μ mol/L was selected based on pilot experiments.
2.4
Statistical analysis
Mann-Whitney U and Wilcoxon signed rank tests in SPSS version 12.0 were used to determine statistical significance. Statistical significance was accepted for P values of <.05.
The International Animal Care and Use Committee at Seoul National University approved the protocol of the animal experiments performed during this study.
2
Methods
2.1
Sensitization of mice and preparation of septal mucosa
2.1.1
Development of mouse model of allergic rhinitis
Thirty-six 4-week-old specific pathogen-free female BALB/c mice weighing 10 to 13 g were used in this study. All mice were treated intraperitoneally (IP) and then challenged intranasally with ovalbumin (OVA) or phosphate-buffered saline (PBS) according to the protocol depicted in Fig. 1. Briefly, group A (n = 13) mice were systemically sensitized with 25 μ g OVA IP dissolved in 300 μ L of PBS containing 2 mg alum (Pierce Biotechnology, Inc, Rockford, IL) as an adjuvant on experimental days 0, 7, and 14. One week after the third IP sensitization, mice were challenged intranasally with OVA for 7 consecutive days. Group B (n = 11) mice were systemically immunized in the same way as group A mice and were challenged intranasally with 20 μ L of PBS for 7 consecutive days. Group C (n = 12) mice were administered PBS IP and intranasally as depicted in Fig. 1.
2.1.2
Observed nasal rubbing frequencies during the first 5 minutes after allergen challenge
Twenty-four hours after the final nasal challenge, 200 μ g OVA was instilled into nasal cavities to provoke allergic symptoms and the numbers of nasal rubbings during the 5 minutes immediately after provocation were counted.
2.1.3
Preparation of nasal septal mucosa
Mice were killed, and both nasal septal mucosae (size range, 2–3 × 3–4 mm) were harvested. Harvested nasal septal mucosae were washed in Dulbecco’s modified Eagle’s medium (DMEM) solution (Dulbecco’s modified Eagle’s medium—Ham’s nutrient F12; Gibco BRL, Grand Island, NY) to remove secretions and blood then placed in the same medium during experiment. Penicillin 10 IU/mL, streptomycin 10 IU/mL, and amphotericin B 0.025 mg/mL were added to the DMEM solution, which was kept at 37°C throughout the procedure.
2.2
Measurement of CBF
We measured CBFs using a digitalized computer analysis system . Harvested septal mucosae were fixed at the center of the base of 35 mm plastic culture dishes using a white gold ring and dental floss. Ciliary beat frequencies were measured using an immersion microscope at ×400 in areas in which cilia were found to be moving actively. A digital charge-coupled camera (XC-HR50; Sony Co, Tokyo, Japan) capable of operating at 60 full frames per second transferred images to a computer equipped with a frame grabber (PCVisionplus; Dalsa Co, St. Laurent, Canada). Ciliary movement was analyzed using software based on fast Fourier transform.
2.3
Design of experiment
2.3.1
Measurement of CBF in vitro within 20 minutes after in vivo nasal allergen challenge
Mucosae were harvested from the septum after euthanizing to measure CBF in vitro, within 20 minutes after in vivo nasal allergen challenge. Ciliary beat frequencies were measured at 0, 5, 10, and 15 minutes.
2.3.2
The effect of histamine on CBF
After measuring CBF for 15 minutes, solutions containing 10 −5 , 10 −3 , or 10 −1 mol/L histamine in DMEM were perfused into cultures to replace the preexisting DMEM solution. Ciliary beat frequency was measured at 0, 5, 10, and 15 minutes after histamine treatment.
2.3.3
Ciliary beat frequency responses after treatment with fexofenadine and DMEM solution
To assess the effect of histamine, CBF was measured after treating histamine-exposed tissue with antihistamine or DMEM solution. Fexofenadine (HanDok Co, Seoul, South Korea) was used as the antihistamine, and a concentration of 100 μ mol/L was selected based on pilot experiments.
2.4
Statistical analysis
Mann-Whitney U and Wilcoxon signed rank tests in SPSS version 12.0 were used to determine statistical significance. Statistical significance was accepted for P values of <.05.
The International Animal Care and Use Committee at Seoul National University approved the protocol of the animal experiments performed during this study.
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