The effect of duration of administration of fluticasone propionate and salmeterol on tracheal responsiveness to ovalbumin and total and differential white blood cell in sensitized guinea pig was examined. Six groups of guinea pigs (
The most important characteristic feature of asthma is a chronic inflammatory disorder of the airway [
The combination of long acting
It was well documented that regular treatment with FP and SM combination resulted in continuous improvement in AHR with maintenance of asthma control in the majority of patients [
However, the effect of combined therapy, administered during, or after sensitization on the asthmatic airway inflammation is not compared yet. Therefore, in this study, the effect of an inhaled corticosteroid, fluticasone propionate and long acting
Guinea pigs were sensitized to (OA) as previously described [
The aerosol was administered in a closed chamber, dimensions 30
Guinea pigs were randomly divided into seven groups ( Control group (group C): receiving Al(OH)3 alone dissolved in 1 mL normal saline and inhaled saline aerosol instead of OA. Treatment and placebo groups A: treated with 250 Treatment and placebo groups B: treated with FP + SM or placebo after sensitization period for 18 days. Treatment and placebo groups C: treated with FP + SM or placebo during sensitization period and evaluated with 18 days delay.
Aerosol FP and placebo were administered using ordinary canister through a modified spacer as previously described [
Description of control, three placebo, and three treated sensitized groups. FP: fluticasone propionate (250
Guinea pigs were sacrificed and their trachea was removed. Each trachea was cut into 10 rings (each containing 2-3 cartilaginous rings). All the rings were then cut open opposite the tracheal muscle and sutured together to form a tracheal chain [
The tissue was then suspended in a 10 mL organ bath (Schuler organ bath type 809, March-Hugstetten, Germany) containing Krebs-Henseliet solution of the following composition (mM): NaCl 120, NaHCO3 25, MgSO4 0.5, KH2PO4 1.2, KCl 4.72, CaCl2 2.5, and dextrose 11. The Krebs solution was maintained at 37°C and gassed with 95% O2 and 5% CO2. The tissue was suspended under an isotonic tension of 1 g and allowed to equilibrate for at least 1 h, while it was washed with Krebs solution every 15 mins.
Responses were measured using the Vernier control-type 850 N sensor with sensitivity range of 0–20 g and resolution of 0.2 mm/turn (Hugo-Sachs Elektronik, Germany) and amplified by amplifier (ML/118 quadribridge amp, March- Hugstetten, Germany) and recorded by powerlab (ML-750, 4 channel recorder, March- Hugstetten, Germany).
The tracheal response to 0.1% solution of OA was measured as follows: 0.25 mL of 4% OA solution was added to the 10 mL organ bath. The degree of tracheal chain contraction was recorded after 2.5 mins and was expressed as proportion (percentage) to contraction obtained by 10
The lungs were lavaged with 2 mL of saline for 5 times (total: 10 mL). One mL of bronchoalveolar lavage (BAL) fluid was stained with Turk solution (1 mL of Glacial Acetic Acid, 1 mL of Gentian Violet Solution 1% and 100 mL Distilled Water) and total WBC was counted in duplicate in a hemocytometer (in a Burker chamber).
The remaining BAL was centrifuged at 2500
The percent improvement in each treatment group was calculated; in cases, the treatment group’s data was greater than that of corresponding placebo as calculated by ([(
All data were quoted as mean ± SEM. Percent improvements were achieved as follows: in cases, the treatment data was greater than that of corresponding placebo, the data obtained in treatment group minus the data obtained in corresponding placebo group was divided by the data obtained in the same placebo group and multiplied by 100 (e.g., [(
Tracheal responses to OA in all placebo groups (A, B, and C) were significantly higher than control group (
Percent improvements in tracheal responses to OA and total and differential count of white blood cells in lung lavage changes in three treatment groups A, B, and C.
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Eosinophil % |
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Values are quoted as mean ± SEM. Percent improvements were achieved as follows. In cases, the treatment data was greater than that of corresponding placebo; the data obtained in treatment group minus the data obtained in corresponding placebo group was divided by the data obtained in the same placebo group and multiplied by 100 (e.g. [(TreatmentA1 − PlaceboA1)/PlaceboA1]100). In cases, the treatment data was lower than that of corresponding placebo; the data obtained in placebo group minus the data obtained in corresponding treatment group was divided by the data obtained in the same treatment group and multiplied by 100 (e.g. [(PlaceboA1 − TreatmentA1)/TreatmentA1]100). Comparisons of the data between three treatment groups were done using one-way analysis of variance (ANOVA) with Tukey-Kramer posttest.
Statistical significance for the difference between the data of group A versus group B: ++
Statistical significance for the difference between the data of group A versus group C: *
Statistical significance for the difference between the data of group B versus group C:
Individual values and mean ± SEM (big symbols with bars) of tracheal response to ovalbumin (OA) in control group and sensitized groups treated with fluticasone + salmeterol and placebo during (A), after (B), and during sensitization period and 18 days delay (C). Tracheal responsiveness to OA was measured by percent contraction obtained by 0.1% solution of OA compared to 10
The mean values of total white blood cell (WBC) as well as the percentage of neutrophils and eosinophils were significantly higher but percentage of lymphocytes and monocytes in BAL of placebo groups A, B and C were significantly lower than those of control group (
Individual values and mean ± SEM (big symbols with bars) of total WBC count (count/mL) (b) in control group and sensitized groups treated with fluticasone + salmeterol and placebo during (A), after (B) and during sensitization period and 18 days delay (C). Tracheal responsiveness to OA was measured by percent contraction obtained by 0.1% solution of OA compared to 10
Mean ± SEM of the percentages of eosinophils (a), neutrophils (b), lymphocytes (c), and monocytes (d) of lung lavage in control group and sensitized groups treated with fluticasone + salmeterol and placebo during (A), after (B), and during sensitization period and 18 days delay (C). Comparison of the data between control and three treated with fluticasone propionate + salmeterol and three placebo groups was done using one-way analysis of variance (ANOVA) with Tukey-Kramer posttest. +:
There were a significant increase in the percentage of monocytes in all treatment groups and a significant decrease in the percentage of eosinophils and further decrease of lymphocytes in treatment group A compared to the corresponding placebo groups (
The mean values of improvement in percentage of eosinophils, lymphocytes, and monocytes in BAL of treatment group A were significantly greater than those of treatment protocol B (
Tracheal response to OA, total WBC count in lung lavage and percentage of eosinophils and neutrophils increased but percentages of, lymphocytes and monocytes decreased in sensitized compared to control animals which were similar to the results of the previous studies [
The most effective agent in asthma therapy is a drug which targets both airway inflammation and smooth muscle dysfunction of the disease. Therefore, the effect of FP and SM on the tracheal responsiveness to OA, total differential WBC count which was administered during and after sensitization and during sensitization with 18 days delay in measurements was examined in this study.
The small effect of FP and SM on percentages of neutrophils in BAL fluid of sensitized animals observed in the present study may suggest that fluticasone propionate prolongs human neutrophil survival by inhibiting apoptosis by having an effect on glucocorticoid receptor [
The effect of FP and SM on the tracheal responsiveness to OA and total and differential WBC count in lung lavage of sensitized guinea pigs seen in the present study, was supported by those who indicated that adding LABA leads to greater improvement in different parameters of asthma than increasing the dose of inhaled corticosteroid (ICS) [
In placebo group A (sensitized animals treated with inhaled placebo during sensitization, PA), most parameters were greater than those of placebo group B (sensitized animals treated with inhaled placebo after sensitization, PB) and placebo group C (sensitized animals treated with inhaled placebo during sensitization but measurement of different parameters were performed 18 days after the end of sensitization period, PC). There was greater sensitization in PA compared to PB and PC. The cause of these findings is perhaps due to an allergen-free period which is supported by the effect of allergen prevention in control of asthma disease [
The novelty of the present study is the evaluation of the effect of FP and SM administration after (examining parameters immediately or with 18 days delay) and during sensitization of guinea pigs. The effect of an allergen free period in animals treated during sensitization on the results of combined therapy was also examined. The improvement in most parameters in treatment group A and C (TA and TC) was greater than group B (TB). The cause of these findings is perhaps due to administration of FP and SM during sensitization which indicates the importance of the early as possible asthma therapy. In fact a permanent loss of pulmonary function or even induced remission in some patients in early treatment of airway inflammation is observed previously [
In a previous study, the effect of FP alone administered during or after sensitization was examined [
While asthma is an airway inflammation disorder [
The results showed that FP and SM had a preventive effect on the tracheal hyperresponsiveness to OA and lung inflammation. This preventive effect was greater when drugs were administered during sensitization. The results indicate that asthma therapy should be started as soon as possible early during the development of airway inflammation in asthmatic patients. The role of an allergen-free environment in the treatment of asthma is also suggested.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was financially supported by the Research Department of Mashhad University of Medical Sciences.