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The aim of the present study was to investigate the clinical importance of inflammatory markers in the pathogenesis of RAO, chronic interstitial pneumopathy and acute to subacute respiratory infections in horses in parallel with the clinical examination scores, TBS and BALF cytology and pulmonary function testing.
To our knowledge, the detection of the concentration of MMP-2 and MMP-9 in BALF by ELISA was the first trial performed in our study and was found to be an easy, accurate and reliable method. In addition, a semi-quantitative densitometry method based on the gelatin zymography bands for estimating the MMP-2 and MMP-9 activity were performed. As the collagen zymography for measuring MMP-8 was impossible to perform; the concentration of MMP-8 was detected in BALF by a fluorescent method.
A total of 64 warmblood horses, admitted to the Equine Clinic, Freie Universität Berlin, were used in the present study. Fifteen clinically healthy horses were used as the healthy standard which had no history of respiratory disease and BALF neutrophils percentage ≤8%. Clinical history, physical examination, endoscopy, radiography of the lung and percentage of neutrophils in BALF and TBS were used as the basis for grouping of diseased horses. RAO-affected horses in exacerbation (n=17), in remission (n=18), horses with chronic interstitial pneumopathy (n=11) and horses with acute to subacute respiratory infection (n=3) are the main disease groups studied.
The total examination score were increased significantly in diseased horses compared to controls. The percentage of neutrophils in BALF and TBS was correlated with the concentration of MMP-2, MMP-8, MMP-9 and IL8 and this support the role of these inflammatory markers in the pathogenesis of lung disease.
MMP-2 concentration was significantly increased in RAO-affected horses in exacerbation (5.21±0.77 ng/ml) and remission (7.67±15.5 ng/ml) compared to clinically healthy horses (2.49±0.83 ng/ml). MMP-9 concentration was significantly increased in RAO-affected horses in exacerbation (433.34±89.05 pg/ml), remission (312.06±23.92 pg/ml) and horses with chronic interstitial pneumopathy (263.2±23.85 pg/ml) compared to healthy horses (176.29±60.22 pg/ml). These results were confirmed by zymography for MMP-2 and MMP-9 activity in 55 horses. MMP-8 concentration was also significantly increased in RAO-affected horses in exacerbation (0.84±1 μmol/l), remission (0.1±0.06 μmol/l) and horses with chronic interstitial pneumopathy (0.02±0.01 μmol/l) compared to healthy individuals (0.01±0.01 μmol/l) as evaluated by fluorescent method. BALF neutrophil percentage showed a high positive correlation with the concentration of MMP-2, MMP-8 and MMP-9. Also, a highly positive correlation was found with the MMP-2 and MMP-9 activity measured by gelatin zymography. These results were suggestive for its role in the pathogenesis of lung disease. In addition, IL-8 was significantly increased in RAO-affected horses in exacerbation and remission.
The observations of the present study provide new insights into the diagnostic potential of various biomarkers in equine airway disease and could be used as an important tool for detecting lung tissue remodeling. Further studies should be focused on the role of TIMPs as a therapeutic approach.