**1. Introduction**

Disorders of the respiratory system, particularly the lower airways, are the most frequently diagnosed conditions in sport horses evaluated for poor performance [1]. The terminology used to describe equine chronic noninfectious small airway disease has further evolved in the last few years with the term "equine asthma" (EA), as new features (functional, anatomical and pathobiological) of this condition are emerging [2,3]. EA is now being recommended to describe horses with chronic respiratory signs ranging in severity from mild to severe: they were previously referred as inflammatory airway disease (IAD) or recurrent airway obstruction (RAO), respectively [3]. RAO is then considered as one of the main features of horses with a severe form of equine asthma (also defined as severe equine asthma syndrome) [2–6], which resembles human asthma in many aspects [3,4].

Horses affected by severe equine asthma syndrome show labored breathing at rest following exposure to specific airborne agents, and reversible and reproducible airway obstruction related to the level of environmental exposures [4]. The exposure to hay and dust leading to heaves is rather a consequence of the human influence on the horses' natural environment. Molds and fungi are indeed common antigens in stables, suggesting that EA is a disease of "domestication". However, horses can develop a similar condition while at pasture, with grass pollen then being the likely triggering factor [7–9].

Clinical exacerbation occurs following exposure of susceptible horses to specific airborne agents, and results in a disease phenotype of varying severity, ranging from exercise intolerance to coughing and severe expiratory dyspnea. As the name implies, the disease is largely reversible, whereby avoidance of the inciting airborne agents results in significant disease remission over time. The cardinal clinical features of severe EA can be due to the underlying airway inflammatory response that underpins the particular functional and pathological features of the disease [10].

Since bronchoalveolar lavage (BAL) by use of fiber-optic endoscopy was first described in horses, cytological and microbiological evaluation of tracheal washes and BAL fluid have become the cornerstones in the diagnosis of respiratory disease alongside clinical and functional examinations. In exacerbation of severe equine asthma, the horses show dyspnea at rest, as revealed by a maximum intrapleural pressure >15 cm H2O caused by bronchoconstriction, mucosal swelling and mucus accumulation [11], and inflammation of the small airways, in which neutrophils exceed 25% in bronchoalveolar lavage fluid (BALF) cytology [2]. The definitive diagnosis of severe equine asthma is usually based on BALF cytology with the presence of lower airway inflammation, characterized by total nucleated cell count with mild increased numbers of neutrophils [12–14] and lymphocytes, and by increased mast cell or eosinophilic counts [15,16]. Therefore, two different cytological phenotypes are recognized in severe equine asthma: a classical neutrophilic phenotype and a paucigranulocytic phenotype. They do not correspond to the histopathological finding observable in the peripheral airway [2] and suggesting a complex role for pulmonary neutrophils in equine asthma pathophysiology. Currently, little is known about the degree and type of BALF inflammation, during the different phases of the disease (asymptomatic, exacerbation and remission phase). Moreover, pathological features of the bronchial mucosa and their value in the diagnosis of severe equine asthma syndrome are poorly characterized. To date, histological findings collected by endoscopic biopsies do not allow differentiating between controls and subjects with severe EA in remission [17,18].

The immunologic background of severe equine asthma remains not fully clarified despite many studies on the pathogenesis [19,20]. However, a positive correlation exists between the intensity of airway hyperreactivity and the quantity of chemical mediators released locally in the lung [21]. Generally, airway inflammation involves activation of a pathogen-specific inflammatory cell, the modulation of gene transcription factors, and release of inflammatory mediators [16]. Commonly accepted airway inflammatory mediators involved in airway disease include histamine, bradykinin, prostaglandin, leukotrienes, platelet-activating factor, and endothelin-1 [20–22]. A tachykinin mediator with a physiological and pathological role in respiratory function is a neuropeptide called neurokinin A (NKA), which is involved in the processes of bronchoconstriction and neurogenic inflammation in asthmatic patients, with potential therapeutic implications using selective NKA receptor antagonists [20]. To the best of our knowledge, only two studies investigated the role of neurokinin-A (NKA) in the horse respiratory tract [20,23], and only one by immunohistochemical methods [20].

Neutrophilic bronchiolitis is one of the main lesions of asthma-affected horse's response to aeroallergens. Within the airways, neutrophils likely contribute to bronchoconstriction, mucus hypersecretion, and pulmonary remodeling by release of pro-inflammatory mediators, including the cytokines, among which interleukins 8 (IL-8) [16]. Determining which cytokines are implicated in the pathogenesis of EA may help in the diagnosis and treatment of this disease.

The aim of the study performed in EA-affected horses in asymptomatic, exacerbation and remission phase, is (1) to verify the diagnostic value of histology and the information deriving from it, using the histological scoring system for endoscopic biopsies, in a possible correlation with the clinical features of the different phases; (2) to analyze the immunohistochemical response for NKA and IL-8 from biopsy samples of lung tissue in subjects undergoing severe EA, in the different phases of experimentally induced disease; and (3) to evaluate the influence of sampling position along the respiratory tract on results.

#### **2. Materials and Methods**
