Platelets in equine recurrent airway obstruction

Platelets contribute to the pathogenesis of human allergic airway disease. The aim of this study was to compare platelet activating factor (PAF)-induced platelet aggregation and thromboxane (Tx) production, plasma Tx and 5-hydroxytryptamine (5-HT) in ponies with recurrent airway obstruction (RAO), an hypersensitivity to inhaled antigens, and normal ponies, before and after antigen exposure. Plasma 5-HT was significantly higher in ponies with RAO but was not further increased by antigen challenge. There was no difference between PAF-induced platelet aggregation or Tx production, or in plasma Tx before or after challenge. These data suggest there may be a difference between platelet 5-HT uptake in RAO and normal ponies but do not provide evidence of platelet activation following antigen exposure.     

Expression of thymic stromal lymphopoietin in equine recurrent airway obstruction

Thymic stromal lymphopoietin (TSLP) is a cytokine involved in lymphocyte development. In humans and mice, TSLP drives the differentiation of T helper 2 (Th2) cells and the development of allergic inflammation. The equine TSLP gene has been previously identified and characterized, but its role in the pathogenesis of equine allergic diseases is not known. Our objective was to assess the expression of TSLP in bronchoalveolar lavage (BAL) cells and in primary bronchial epithelial cells (BEC) isolated from horses with recurrent airway obstruction (RAO). RNA was isolated from BAL cells sampled from clinical cases of RAO (n=8) and from control horses (n=12). Furthermore, BAL samples were taken from an additional group of 8 RAO-susceptible and 8 control horses when on pasture (remission) and after 30 days of exposure to moldy hay (exacerbation). In order to study epithelial cells as a potential source of TSLP, cultures of primary bronchial epithelial cells (BEC) were established from 6 RAO-affected and 6 healthy horses and stimulated in vitro with hay dust solution (HDS). Expression of TSLP mRNA was assessed by quantitative real-time RT-PCR (qPCR). Clinical RAO-cases had higher TSLP expression in BAL than control horses (p<0.05). In an experimental group of horses there was no difference between healthy and susceptible horses in remission, whereas after 30-day experimental exposure to moldy hay, all susceptible horses upregulated TSLP expression in BAL (p=0.008, average 6.36-fold increase), whereas in healthy horses there was no significant increase in TSLP expression. BEC generated both from healthy and RAO-affected horses strongly upregulated TSLP expression after 6 h stimulation with HDS, which identifies epithelial cells as potential sources of TSLP in RAO. Finding of increased TSLP expression by BAL cells of RAO-affected horses is in agreement with the contribution of Th2-driven allergic inflammation in the pathogenesis of RAO.     

Temporal regulation of cytokine mRNA expression in equine recurrent airway obstruction

Acute and chronic inflammation of the airway remains an important health problem for equids. "Heaves" or recurrent airway obstruction (RAO) remains one of the most commonly diagnosed conditions affecting the lung of older horses in Europe and the United States. The typical clinical signs of RAO include non-productive coughing, serous nasal discharge, labored expiratory effort, and flaring of the nostrils. Auscultation of the lungs of the affected horse often reveals abnormal respiratory sounds, described as crackles and wheezes, throughout the area of the lung field. These clinical signs occur secondary to an inflammatory response that results in bronchospasm, excessive mucus production and airway obstruction. This inflammatory response is characterized by the presence of excessive mucus and inflammatory cells, primarily neutrophils, in the small airways. Most evidence suggests that RAO is the result of a pulmonary hypersensitivity to inhaled antigens. Exposure of affected horses to hay dust, pollens, and mold spores leads to neutrophil accumulation in the lung and bronchospasm. The identification of allergen-specific IgE in bronchoalveolar lavage (BAL) fluid and sera of affected horses supports the involvement of a late phase, IgE-mediated, hypersensitivity reaction in the pathogenesis of equine RAO. The production of IgE antibodies is regulated by the cytokines IL-4 and IL-13. Using a quantitative PCR method we have reported that horses with RAO exhibit a modified Type 2 cytokine response characterized by the production of IL-4 and IL-13 mRNA, but not IL-5 mRNA in BAL cells. Interferon-gamma mRNA was also elevated, suggesting a mixed response. While these results are consistent with equine RAO being the result of an aberrant Type 2 cytokine response to inhaled allergens, others have failed to find any evidence of elevated Type 2 cytokine mRNA in BAL from horses with "heaves". It is likely that these disparate results could be the result of differences in the clinical stage of the affected animals or the timing of sample collection. Here, we report a diverse pattern of cytokine gene expression when sampling a group of affected horses over a period of time.     

A region on equine chromosome 13 is linked to recurrent airway obstruction in horses

REASONS FOR STUDY: Equine recurrent airway obstruction (RAO) is probably dependent on a complex interaction of genetic and environmental factors and shares many characteristic features with human asthma. Interleukin 4 receptor a chain (IL4RA) is a candidate gene because of its role in the development of human asthma, confirmation of this association is therefore required. METHODS: The equine BAC clone containing the IL4RA gene was localised to ECA13q13 by the FISH method. Microsatellite markers in this region were investigated for possible association and linkage with RAO in 2 large Warmblood halfsib families. Based on a history of clinical signs (coughing, nasal discharge, abnormal breathing and poor performance), horses were classified in a horse owner assessed respiratory signs index (HOARSI 1-4: from healthy, mild, moderate to severe signs). Four microsatellite markers (AHT133, LEX041, VHL47, ASB037) were analysed in the offspring of Sire 1 (48 unaffected HOARSI 1 vs. 59 affected HOARSI 2-4) and Sire 2 (35 HOARSI 1 vs. 50 HOARSI 2-4), age 07 years. RESULTS: For both sires haplotypes could be established in the order AHT133-LEXO47-VHL47-ASB37. The distances in this order were estimated to be 2.9, 0.9 and 2.3 centiMorgans, respectively. Haplotype association with mild to severe clinical signs of chronic lower airway disease (HOARSI 2-4) was significant in the offspring of Sire 1 (P = 0.026) but not significant for the offspring of Sire 2 (P = 0.32). Linkage analysis showed the ECA13q13 region containing IL4RA to be linked to equine chronic lower airway disease in one family (P<0.01), but not in the second family. CONCLUSIONS: This supports a genetic background for equine RAO and indicates that IL4RA is a candidate gene with possible locus heterogeneity for this disease. POTENTIAL RELEVANCE: Identification of major genes for RAO may provide a basis for breeding and individual prevention for this important disease.     

Effects of a MAPK p38 inhibitor on lung function and airway inflammation in equine recurrent airway obstruction

Reasons for performing study: It has been suggested that many of the beneficial effects of corticosteroids are mediated through mitogen‐activated protein kinase (MAPK) p38 inhibition. Objective: To investigate the efficacy of the MAPK p38 inhibitor compound MRL‐EQ1 to either prevent (Phase 1) or treat (Phase 2) recurrent airway obstruction (RAO) in horses. Methods: MRL‐EQ1 was administered i.v. at a dosage of 0.75‐1.5 mg/kg bwt q. 12 h. In Phase 1, susceptible horses in clinical remission were divided into 2 groups (n = 5/group), based on historical values of respiratory mechanics. All horses were entered in the study in pairs (one control, one treated horse) and exposed to the same environmental challenge (stabling, mouldy hay and dusty conditions). The treatment group received MRL‐EQ1 for 14 days while the control horses were untreated during the same period. In Phase 2, affected horses were ranked by severity of respiratory dysfunction and split randomly into either dexamethasone or MRL‐EQ1 treatment groups (n = 5/group). Bronchoalveolar lavage fluid, respiratory mechanic measurements, MRL‐EQ1 plasma concentration and tumour necrosis factor (TNF) whole blood activity were evaluated sequentially. Results: In Phase 1, MRL‐EQ1 did not prevent the occurrence of clinical signs and pulmonary inflammation. However, treatment was associated with a reduction in severity and a delay in the onset of signs and a reduction in pulmonary neutrophilia. In Phase 2, plasma concentrations achieved resulted in ex vivo suppression of lipopolysaccharide‐induced TNF production in equine blood. MRL‐EQ1 did not improve airway inflammation or lung function and was associated in a dose dependent manner with behavioural (depression, excitability) and blood changes (neutrophilia, increased serum muscle enzyme concentrations). Conclusions: Inhibition of p38 in the horse was partially effective in reducing clinical signs and airway inflammation when administered prior to, but not during clinical exacerbation in RAO. Potential relevance: Inhibitors of p38 MAPK with a better toxicity profile may be effective in the prevention or treatment of RAO.     

Genetics of recurrent airway obstruction (RAO)

Recurrent airway obstruction (RAO) is a multifactorial and polygenic disease. Affected horses are typically 7 years of age or older and show exercise intolerance, increased breathing effort, coughing, airway neutrophilia, mucus accumulation and hyperreactivity as well as cholinergic bronchospasm. The environmental factors responsible are predominantly allergens and irritants in haydust, but the immunological mechanisms underlying RAO are still unclear. Several studies have demonstrated a familiar predisposition for RAO and it is now proven that the disease has a genetic basis. In offspring, the risk of developing RAO is 3-fold increased when one parent is affected and increases to almost 5-fold when both parents have RAO. Segregation analysis in two high-prevalence families demonstrated a high heritability and a complex inheritance with several major genes. A whole genomescan showed chromosome-wide significant linkage of seven chromosomal regions with RAO. Of the microsatellites, which were located near atopy candidate genes, those in a region of chromosome 13 harboring the IL4R gene were strongly associated with the RAO phenotype in the offspring of one RAO-affected stallion. Furthermore, IgE-levels are influenced by hereditary factors in the horse, and we have evidence that RAO-affected offspring of the same stallion have increased levels of specific IgE against moldspore allergens. The identification of genetic markers and ultimately of the responsible genes will not only allow for an improved prophylaxis, i.e. early identification of susceptible individuals and avoidance of high-risk matings, but also improve our ability to find new therapeutic targets and to optimize existing treatments.     

Cardiovascular effects of acute pulmonary obstruction in horses with recurrent airway obstruction

BACKGROUND: Recurrent airway obstruction (RAO) is common in horses. Although pulmonary artery (PA) pressure increases during RAO, cardiac function in horses with RAO has received limited attention. HYPOTHESIS: The purpose of this study was to noninvasively determine the cardiovascular effects of acute pulmonary obstruction (APO) in horses with RAO and their reversibility. ANIMALS: Five geldings with RAO, inducible by exposure to moldy hay, were studied. METHODS: Pulmonary mechanics, echocardiography, serum troponin I concentrations, arterial blood gases, and hematocrit were obtained before and after 7 days of APO. Heart rate, PA diameter and flow characteristics, right and left ventricular luminal dimensions and wall thicknesses, global cardiac performance, and evidence of myocardial damage were evaluated. Pulmonary mechanics and echocardiography were reevaluated during remission. RRESULTS: Severe, transient APO did not induce chronic cor pulmonale in horses, because cardiac anatomy and function were normal between episodes. An acute episode of APO produced anatomical and functional cardiac changes in both the right and left heart (including increased PA diameter, abnormal septal motion, and decreased left ventricular diameter and estimated stroke volume), possibly because of the development of pulmonary hypertension, without apparent myocardial damage. The decrease in stroke volume was offset by the increase in heart rate. CONCLUSIONS AND CLINICAL IMPORTANCE: With APO of 7 days' duration, cardiovascular abnormalities and the functional airway changes that produce them are reversible when the offending allergens are removed.     

Effects of xylazine on airway function in ponies with recurrent airway obstruction.

The effect of IV administration of the alpha 2-adrenoceptor agonist xylazine hydrochloride (0.5 mg/kg of body weight) was examined in ponies with recurrent obstructive pulmonary disease, commonly called heaves. Six ponies with the disease (principals) were studied during clinical remission and during an acute attack of airway obstruction precipitated by stabling and feeding of dusty hay. Six control ponies were also studied. In principal ponies with airway obstruction, xylazine administration significantly (P < 0.05) decreased pulmonary resistance and increased dynamic compliance, but did not affect PaO2 or PaCO2. The alpha 2-antagonist yohimbine blocked the pulmonary effects of xylazine. Administration of saline solution was without effect in both groups of ponies at all periods and xylazine did not have effect in controls or in principals in clinical remission.     

Coughing,mucus accumulation,airway obstruction,and airway inflammation in control horses and horses affected with recurrent airway obstruction

OBJECTIVE: To investigate relationships between cough frequency and mucus accumulation, airway obstruction, and airway inflammation and to determine effects of dexamethasone on coughing and mucus score. ANIMALS: 13 horses with recurrent airway obstruction (RAO) and 6 control horses. PROCEDURE: 6 RAO-affected and 6 control horses were stabled for 3 days. Coughing was counted for 4 hours before and on each day horses were stabled. Before and on day 3 of stabling, tracheal mucus accumulation was scored, airway obstruction was assessed via maximal change in pleural pressure (deltaPpl(max)), and airway inflammation was evaluated by use of cytologic examination of bronchoalveolar lavage fluid (BALF). Effects of dexamethasone (0.1 mg/kg, IV, q 24 h for 7 days) were determined in 12 RAO-affected horses. RESULTS: To assess frequency, coughing had to be counted for 1 hour. In RAO-affected horses, stabling was associated with increases in cough frequency, mucus score, and deltaPpl(max). Control horses coughed transiently when first stabled. In RAO-affected horses, coughing was correlated with deltaPpl(max), mucus score, and airway inflammation and was a sensitive and specific indicator of deltaPpl(max) > 6 cm H2O, mucus score > 1.0, and > 100 neutrophils/microL and > 20% neutrophils in BALF Dexamethasone reduced cough frequency, mucus score, and deltaPpl(max), but BALF neutrophil count remained increased. CONCLUSIONS AND CLINICAL RELEVANCE: Because of its sporadic nature, coughing cannot be assessed accurately by counting during brief periods. In RAO-affected horses, coughing is an indicator of airway inflammation and obstruction. Corticosteroid treatment reduces cough frequency concurrently with reductions in deltaPpl(max) and mucus accumulation in RAO-affected horses.     

Immunological characterization of the equine airway epithelium and of a primary equine airway epithelial cell culture model

Our understanding of innate immunity within the equine respiratory tract is limited despite growing evidence for its key role in both the immediate defense and the shaping of downstream adaptive immune responses to respiratory disease. As the first interface to undergo pathogen invasion, the respiratory epithelium is a key player in these early events and our goal was to examine the innate immune characteristics of equine respiratory epithelia and compare them to an in vitro equine respiratory epithelial cell model cultured at the air-fluid interface (AFI). Respiratory epithelial tissues, isolated epithelial cells, and four-week old cultured differentiated airway epithelial cells collected from five locations of the equine respiratory tract were examined for the expression of toll-like receptors (TLRs) and host defense peptides (HDPs) using conventional polymerase chain reaction (PCR). Cultured, differentiated, respiratory epithelial cells and freshly isolated respiratory epithelial cells were also examined for the expression of TLR3, TLR9 and major histocompatibility complex (MHC) class I and class II using fluorescence-activated cell sorting (FACS) analysis. In addition, cytokine and chemokine profiles from respiratory epithelial tissues, freshly isolated respiratory epithelial cells, and cultured, differentiated, epithelial cells from the upper respiratory tract were examined using real-time PCR. We found that respiratory epithelial tissues and isolated epithelial cells expressed TLRs 1-4 and 6-10 as well as HDPs, MxA, 2'5' OAS, β-defensin-1, and lactoferrin. In contrast, epithelial cells cultured at the AFI expressed TLRs 1-4 and 6 and 7 as well as MxA, 2'5' OAS, β-defensin-1, but had lost expression of TLRs 8-10 and lactoferrin. In addition, MHC-I and MHC-II surface expression decreased in epithelial cells cultured at the AFI compared to isolated epithelial cells whereas TLR3 and TLR9 were expressed at similar levels. Lastly, we found that equine respiratory epithelial cells express an array of pro-inflammatory, antiviral and regulatory cytokines and that after four weeks of in vitro growth conditions, equine respiratory epithelial cells cultured at the AFI retained expression of GM-CSF, IL-10, IL-8, TGF-β, TNF-α, and IL-6. In summary, we describe the development of an in vitro equine respiratory epithelial cell culture model that is morphologically similar to the equine airway epithelium and retains several key immunological properties. In the future this model will be a used to study equine respiratory viral pathogenesis and cell-to-cell interactions.     

Development of equine upper airway fluid mechanics model for Thoroughbred racehorses

REASON FOR PERFORMING STUDY: Computational fluid dynamics (CFD) models provide the means to evaluate airflow in the upper airways without requiring in vivo experiments. HYPOTHESIS: The physiological conditions of a Thoroughbred racehorse's upper airway during exercise could be simulated. Methods: Computed tomography scanned images of a 3-year-old intact male Thoroughbred racehorse cadaver were used to simulate in vivo geometry. Airway pressure traces from a live Thoroughbred horse, during exercise was used to set the boundary condition. Fluid-flow equations were solved for turbulent flow in the airway during inspiratory and expiratory phases. The wall pressure turbulent kinetic energy and velocity distributions were studied at different cross-sections along the airway. This provided insight into the general flow pattern and helped identify regions susceptible to dynamic collapse. RESULTS: The airflow velocity and static tracheal pressure were comparable to data of horses exercising on a high-speed treadmill reported in recent literature. The cross-sectional area of the fully dilated rima glottidis was 7% greater than the trachea. During inspiration, the area of highest turbulence (i.e. kinetic energy) was in the larynx, the rostral aspect of the nasopharynx was subjected to the most negative wall pressure and the highest airflow velocity is more caudal on the ventral aspect of the nasopharynx (i.e. the soft palate). During exhalation, the area of highest turbulence was in the rostral and mid-nasopharynx, the maximum positive pressure was observed at the caudal aspect of the soft palate and the highest airflow velocity at the front of the nasopharynx. CONCLUSIONS AND CLINICAL RELEVANCE: In the equine upper airway collapsible area, the floor of the rostral aspect of the nasopharynx is subjected to the most significant collapsing pressure with high average turbulent kinetic during inhalation, which may lead to palatal instability and explain the high prevalence of dorsal displacement of the soft palate (DDSP) in racehorses. Maximal abduction of the arytenoid cartilage may not be needed for optimal performance, since the trachea cross-sectional area is 7% smaller than the rima glottidis.     

The activity and expression of chitinase in the equine lung and its activity in normal horses and animals with recurrent airway obstruction

Recurrent airway obstruction (RAO) is a chronic inflammatory condition in equine lung, which may share a common immunological basis with human asthma, in which dysregulated Th2 responses occur. Mammals express chitinases and chitinase-like proteins, two of which are active enzymes, chitotriosidase and acidic mammalian chitinase (AMCase). Both enzymes are upregulated in a range of inflammatory conditions, including asthma. We investigated the activity of chitinase in bronchoalveolar lavage fluid from horses with and without RAO in response to organic dust challenges. No significant differences were found in activity, although in one study RAO animals had elevated chitinase activity that fell short of statistical significance. The pH optimum and pH lability of the activity was consistent with the presence of chitotriosidase. RT-PCR amplification of the mRNA encoding chitotriosidase and AMCase in normal equine lung showed that chitotriosidase, but not AMCase, is expressed in trachea, bronchi, and peripheral lung tissue. The gene for chitotriosidase was identified from the Equus caballus (horse) genome 1.1 database and its similarity to the same genes from other species was determined. The results of this study indicate that the involvement of chitotriosidase in RAO is uncertain.     

Equine recurrent airway obstruction does not alter airway muscarinic acetylcholine receptor expression and subtype distribution

In recurrent airway obstruction (RAO) or heaves, bronchospasm has been attributed to enhanced cholinergic activity. However, the expression and function of muscarinic acetylcholine receptors (mAChR) and their signaling components are not yet known. Thus, we examined the expression, subtype distribution and postreceptor signaling pathways of mAChR in the peripheral lung, bronchial and tracheal epithelia with the underlying smooth muscle from nine horses with RAO and 11 healthy control horses. In RAO horses, no significant segment-dependent alteration in mAChR density and subtype distribution (assessed by [N-methyl-3H]-scopolamine binding; ([3H]-NMS)), was found, except a trend in receptor down-regulation in some peripheral parts of the lung. The total number of high mAChR binding sites (assessed by carbachol-displacement experiments in the presence or absence of guanosine 5'-triphosphate) was not changed in RAO, suggesting that the functional coupling of mAChR to the corresponding G-proteins is intact. The M2-mediated inhibition of adenylate cyclase (AC) as well as the M3-receptor-G(q/11)-phospholipase C (PLC) activity was not different between RAO and control airway tissues. In conclusion, in equine RAO airways, mAChR expression and function were not altered, and thus appear not to account for the enhanced cholinergic activity in RAO.     

Efficacy of salmeterol xinafoate in horses with recurrent airway obstruction.

OBJECTIVE: To determine the onset, magnitude, and duration of bronchodilation after administration of aerosolized salmeterol xinafoate in horses with recurrent airway obstruction. DESIGN: Randomized controlled study ANIMALS: 6 horses with recurrent airway obstruction. Procedure Horses received aerosolized salmeterol (210 microg) or no treatment, using a crossover design. Salmeterol was administered, using a mask designed for aerosol delivery in horses. Subjective rating of airway obstruction (RAO), maximal change in pleural pressure (deltaPplmax), and pulmonary resistance (RL) were determined at baseline; 5, 15, and 30 minutes; and 1, 2, 4, 6, 8, 10, and 12 hours after administration of salmeterol and in horses that did not receive treatment. RESULTS: The deltaPpl and RL were improved 15 minutes through 6 hours after administration of salmeterol, compared with values obtained from horses receiving no treatment. The RAO was improved 15 minutes through 2 hours after administration of salmeterol. The maximal response to salmeterol was evident 30 to 60 minutes after administration and was characterized by a 59 + 19% decrease in deltaPpl and a 56 +/- 13% decrease in RL. The deltaPpl and RL were not different from baseline values 8 hours after salmeterol administration. CONCLUSIONS AND CLINICAL RELEVANCE: Duration of action of salmeterol in these horses was approximately 6 hours. Maximal bronchodilation was somewhat delayed (30 to 60 minutes), and the magnitude of response was similar to that of short-acting beta2-adrenergic agonists. Salmeterol provides moderately sustained bronchodilation in horses with recurrent airway obstruction and may be an effective drug for long-term control of this condition.     

Neurokinin receptors in recurrent airway obstruction: A comparative study of affected and unaffected horses

The purpose of the study was to compare in vitro airway responses to neurokinin A & B (NKA and NKB) and expression of NK-2 receptors in airways of horses affected and unaffected with recurrent airway obstruction (RAO). Neurokinin-A, an inflammatory mediator belonging to the tachykinin family of neuropeptides, causes bronchoconstriction by binding to NK-2 receptors. Neurokinin-B is a lesser-known neuropeptide that acts on NK-3 receptors. Horses were placed into RAO-affected and RAO-unaffected groups based on their history, clinical scoring, and pulmonary function testing. Lung tissue from each lobe was collected for immunohistochemical staining for NK-2 receptors. Cumulative concentration-response relationships were determined on bronchial rings (4-mm wide) collected and prepared from the right diaphragmatic lung lobe to graded concentrations (half log molar concentrations 10⁻⁷M to 10⁻⁴M) of NKA and NKB. The results showed that NKA caused significantly greater contraction than NKB in both groups. In RAO-affected horses, both agents produced significantly greater bronchial contractions than those in the RAO-unaffected horses. Immunohistochemical staining showed that the overall NK-2 receptor distribution was significantly increased in bronchial epithelium and smooth muscles of bronchi and pulmonary vessels of RAO-affected than RAO-unaffected horses. The findings indicate that NK-2 receptors are up-regulated in RAO, suggesting that NK-2 receptor antagonists may have some therapeutic value in controlling the progression of airway hyperreactivity in horses affected with RAO.     

Lung function and airway cytologic profiles in horses with recurrent airway obstruction maintained in low-dust environments

BACKGROUND: The effects of long-term environmental management on airway obstruction and inflammation in horses with recurrent airway obstruction (RAO) are unknown. HYPOTHESIS: Horses with RAO maintained in low-dust environments have persistent airway obstruction and neutrophilic inflammation. ANIMALS: Study horses were treated for RAO and then maintained in low-dust environments with no medical management. Horses were classified into 3 groups by years after diagnosis: 1 year (time 1, n = 9), 2-3 years (time 2, n = 7), and 5-6 years (time 3, n = 8). The comparison groups were age-matched healthy horses. METHODS: In this cross-sectional study, a clinical examination was performed, and the clinical score was calculated. Standard lung function, forced expiratory maneuvers, and the cytology of bronchoalveolar lavage fluid (BALF) were evaluated. RESULTS: The clinical scores of the RAO horses were higher than those of the non-RAO horses at time 2 (P = .018). Standard lung function data were not different between the groups at any time point. The forced expiratory flow between 75-95% of exhaled vital capacity was lower in RAO horses than in non-RAO horses at all time points (P < .02), indicating persistent peripheral airway obstruction. Cytologic evaluation of BALF revealed no difference in total nucleated cell numbers or differential cell counts between RAO and non-RAO horses at any time point. CONCLUSIONS AND CLINICAL IMPORTANCE: The peripheral airway obstruction detected in horses with RAO maintained in low-dust environments likely is due to irreversible airway remodeling but is not associated with cytologic evidence of airway inflammation.