*Article*

#### **Immunohistochemical Expression of Neurokinin-A and Interleukin-8 in the Bronchial Epithelium of Horses with Severe Equine Asthma Syndrome during Asymptomatic, Exacerbation, and Remission Phase**

**Maria Morini 1,\*, Angelo Peli 1, Riccardo Rinnovati 1, Giuseppe Magazzù 2, Noemi Romagnoli 1, Alessandro Spadari 1 and Marco Pietra 1**


**Citation:** Morini, M.; Peli, A.; Rinnovati, R.; Magazzù, G.; Romagnoli, N.; Spadari, A.; Pietra, M. Immunohistochemical Expression of Neurokinin-A and Interleukin-8 in the Bronchial Epithelium of Horses with Severe Equine Asthma Syndrome during Asymptomatic, Exacerbation, and Remission Phase. *Animals* **2021**, *11*, 1376. https:// doi.org/10.3390/ani11051376

Academic Editors: Alejandro Suárez-Bonnet and Gustavo A. Ramírez Rivero

Received: 7 April 2021 Accepted: 6 May 2021 Published: 12 May 2021

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**Simple Summary:** Severe equine asthma (EA) syndrome, formerly termed Recurrent Airway Obstruction (RAO) or heaves, is one of the most common respiratory diseases in adult horses and a frequent cause of poor equine performance. The affected animals may show periods of clinical remission followed by periods of exacerbation over months to years. Therefore, the aim of the present study was to investigate the histological features, and the Neurokinin-A (NKA) and Interleukin-8 (IL-8) immunoreactivity on bronchoscopic biopsies in horses, obtained during different phases of the disease (asymptomatic, exacerbation and remission). Histological samples of EA-affected horses appeared significantly different from those of non-EA-affected horses (control group) throughout the experimental phase, from inclusion to exacerbation and remission, and intensity of NKA immunopositivity of horses with severe EA was significantly higher than that of control horses in late exacerbation and in remission phase. No significant difference between horses with severe EA in each phase and control horses was noticed for IL-8 immunoreactivity. Moreover, no influence of bronchial sampling position on histological and immunohistochemistry results was found, and it suggests that bronchial structural and functional modification during severe equine asthma tends to be distributed homogeneously throughout the respiratory tree.

**Abstract:** Severe equine asthma (EA) syndrome is a chronic obstructive disease characterized by exaggerated contraction, inflammation, and structural alteration of the airways in adult horses, when exposed to airborne molds and particulate material. However, little is known about the relationship between the degree and type of inflammation on one hand, and the severity of the disease and the response to treatment on the other. Furthermore, to date, very few studies evaluate the diagnostic value of histology and immunohistochemical features of endoscopic biopsies on subjects with severe equine asthma. To investigate the expression of two inflammatory markers (NKA and IL-8) before, during, and after the exacerbation of severe EA, a histological and immunohistochemical study was carried out on a series of biopsy samples collected by bronchoscopy from six EA-affected horses subjected to process exacerbation through environmental stimuli and then to pharmacological treatment. The application of a histological biopsy scoring system revealed a significant difference between control cases and the EA-affected horses in all experimental phases (asymptomatic, early exacerbation phase, late exacerbation phase, and remission phase). For immunohistochemistry (IHC), only the intensity of NKA positivity increases significantly between control horses and the EA horses at late exacerbation and remission phases. In EA-affected horses, a difference was detected by comparing histology between asymptomatic and remission phase, meanwhile, NKA and IL-8 showed no differences between the experimental phases. Based on these results we can assert that: (1) The endoscopic biopsies generate reliable and homogeneous samples in the entire bronchial tree; (2) the clinical improvement associated with treatment is characterized by a significant worsening of the histological findings; and (3) the NKA immunopositivity seems to increase significantly rather

than decrease, as one would have expected, after pharmacological treatment. Further studies are necessary both to implement the number of samples and to use other markers of inflammation to characterize the potential role of cytokines in the diagnosis and therapeutic approach of severe equine asthma.

**Keywords:** respiratory tract; horse; severe equine asthma; immunohistochemistry; IL-8; NKA
