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Equine dental diseases are often underdiagnosed and their signs inadequately reported. Many horse owners have difficulties in recognizing pain-related behavioral signs and in associating them with dental pain. Our objective was to determine what type and degree of dental findings may cause behavioral signs associated with dental pain. In this cross-sectional study, dental examination was performed on 183 adult horses and cheek tooth findings were scored. Owners filled in an internet-based questionnaire including 35 questions concerning eating behavior, bit behavior, and general behavior of the horse. Descriptive statistics and logistic regression analyses were performed. Broadened or darkened fissures [odds ratio (OR) 2.4, 95% confidence interval (CI) 1.04–5.7), complicated fractures (OR 2.3, CI 1.01–5.2) and secondary dentine defects of at least the second degree (OR 3.1, CI 1.2–7.7) were associated with the expression of at least five behavioral signs in the univariable binomial logistic regression analyses. Horses with at least one of these potentially painful cheek tooth findings expressed more signs related to eating behavior, bit behavior, and general behavior than did the other horses. The results suggest that cheek tooth findings indicated by this study as being potentially painful, i.e. broadened or darkened fissures, complicated fractures and secondary dentine defects of at least the second degree, may require intervention, particularly if the horse expresses any behavioral signs that might be related to dental pain.

OBJECTIVE To examine whether expression of extracellular matrix metalloproteinase inducer (EMMPRIN) can be detected in equine lungs and whether it correlates with matrix metalloproteinase (MMP)-2 and -9 expression in bronchoalveolar lavage fluid (BALF) of horses with chronic inflammation of the lungs (ie, lower airway inflammation [LAI]). ANIMALS 29 horses with signs of chronic respiratory tract disease, which were classified as the LAI (n = 17) and LAI with respiratory distress (RDLAI [12]) groups, and 15 control horses. PROCEDURES BALF, tracheal aspirate, and blood samples were obtained, and EMMPRIN expression was determined from BALF cells and RBCs by use of western blotting. Activities of MMP-2 and -9 were determined with zymography. RESULTS Expression of EMMPRIN protein was identified in BALF cells of all horses. Expression of EMMPRIN protein was highest for the RDLAI group and was correlated with MMP-2 and -9 protein expression, MMP-9 gelatinolytic activity, and airway neutrophilia. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that EMMPRIN was involved in the pathophysiologic processes of asthma in horses. However, additional studies of horses and other species are warranted to elucidate the regulation of EMMPRIN expression in asthmatic lungs.

Pneumonia is one of the potential complications of general anaesthesia in horses. Anaesthesia is known to increase neutrophils in bronchoalveolar lavage fluid (BALF) of horses after lateral recumbency, but studies after dorsal recumbency are lacking. Our primary aim was to determine when lung inflammation reaches its maximum and how rapidly BALF cytology returns to baseline after anaesthesia in dorsal recumbency. A secondary aim was to investigate the possible effect of vatinoxan, a novel drug, on the BALF cytology results. Six healthy experimental horses were enrolled in this observational crossover study. The horses were subject to repeated BALF and blood sampling for 7 days after general anaesthesia with two treatment protocols, and without anaesthesia (control). During the two treatments, the horses received either medetomidine-vatinoxan or medetomidine-placebo as premedication, and anaesthesia was induced with ketamine-midazolam and maintained with isoflurane for 1 h in dorsal recumbency. The differences in BALF and blood variables between the two anaesthesia protocols and control were analysed with repeated measures analysis of variance models. In this study, anaesthesia in dorsal recumbency resulted in no clinically relevant changes in airway cytology that could be differentiated from the effect of repeated BALF sampling. No differences in BALF matrix metalloproteinase gelatinolytic activity could be detected between the two treatments or the control series. Marked increase in serum amyloid A was detected in some animals. Vatinoxan as premedication did not consistently affect lung cytology or blood inflammatory markers after anaesthesia. (C) 2019 The Author(s). Published by Elsevier Ltd.

Most equine lower respiratory diseases present as increased airway neutrophilia, which can be detected in tracheal wash (TW) or bronchoalveolar lavage fluid (BALE) cytology samples. The aim was to compare the TW and BALF results in a population of client-owned horses with and without clinical respiratory disease signs. A secondary aim was to determine the sensitivity (Se) and specificity (Sp) of TW and BALF neutrophilia in detecting respiratory disease. The cutoff values for neutrophils were also evaluated. Retrospective data from 154 horses of various breeds that had been subject to both TW and bronchoalveolar lavage (BAL) sampling at rest during 2009-2015 were used. The horses were divided into three groups based on the presenting signs, physical examination, and endoscopy mucus score. Neutrophil counts of >20% in TW and >5% in BAL were considered abnormal. Cytology results between groups, correlations between 1W and BALF cell types, and tracheal mucus score were analyzed. Two graph receiving operating characteristic (ROC) curves of the neutrophil percentage values of TW and BALF were created to determine the optimal cutoff values and to calculate the diagnostic Se and Sp for diagnosing airway inflammation in horses with and without clinical respiratory signs. The Se and Sp of TW and BALF neutrophil percentages were further estimated using a two-test one-population Bayesian latent class model. The two tests showed substantial agreement, and only 17.5% of the horses were classified differently (healthy vs. diseased). The neutrophil percentage was found to correlate between TW and BALF. The Se and Sp of TW were generally higher than for BAL when estimated with area under the curve or Bayesian model. Cutoff values of 17.7% for TW and 7% for BALF were indicated by the ROCs. We conclude that TW is a more sensitive and specific method in our patient population. We suggest that the current neutrophil cutoff values of 20% for 1W and 5% for BALE would still be appropriate to use in clinical diagnosis of airway inflammation. However, further studies with other cell types and in other populations are warranted to determine the best sampling method for individual horses.

Non-infectious inflammation of the lower airways, the main theme of this thesis, is commonly observed regardless of the breed, age, or use of the horse. Due to similarities with human asthma regarding clinical presentation and several pathophysiological aspects, non- infectious chronic lower airway inflammation in horses is currently named equine asthma (previously inflammatory airway disease or recurrent airway obstruction, depending on the severity). Long-term exposure to organic dust and moulds in stable air is thought to be the most important predisposing factor for the disease. Equine asthma is characterized by airway hyperresponsiveness and mucus accumulation, severity-related airway remodelling, impaired lung gas exchange, and increased quantity of airway inflammatory cells. These changes lead to clinical signs, including chronic cough and nasal discharge, poor performance, and in a severe form of equine asthma, increased respiratory effort at rest corresponding to an “asthma attack” in humans. Despite active research over the years, the aetiology and pathophysiology of equine asthma remain incompletely defined. In this thesis, the role of a transmembrane protein extracellular matrix metalloproteinase inducer (EMMPRIN), a potential new biomarker in equine asthma, and its relationship to matrix metalloproteinases (MMPs) were investigated. In Study I, the respiratory secretions of 29 horses with asthma and 15 asymptomatic controls were examined. Expression of EMMPRIN protein was identified in all horses in bronchoalveolar lavage fluid (BALF) cells, and the expression was highest for the most substantially affected horses with severe asthma and was correlated with MMP-2 and -9 protein expression, MMP-9 gelatinolytic activity, and airway neutrophilia. This is the first study to describe the relationship of EMMPRIN and MMPs in patients of any species clinically affected with asthma, and the results suggest that EMMPRIN is involved in the pathophysiologic processes of asthma in horses. EMMPRIN as a marker of inflammation provides interesting possibilities for further research of horses and other species. The second part of this thesis concentrated on two common diagnostic tools used to assess lower airway diseases in horses. The diagnosis of lower airway inflammation, including asthma, is often aided by the analysis of cytological samples obtained from the trachea (tracheal wash; TW) or from the lungs at alveolar level (bronchoalveolar lavage; BAL). There is considerable variance in the diagnostic methods of equine lower airway diseases between facilities and clinicians, and a true gold standard for diagnosing airway inflammation is lacking. In most previous studies, the cytology of TW and BAL samples has been considered to correlate poorly and BAL has been deemed a superior diagnostic tool in most cases of equine lower airway disease, but sensitivity and specificity have not been evaluated. Moreover, the conventionally used cut-off values for cytology have been largely based on observational studies and expert opinion, and recently, higher values have been proposed. The first aim of Study II of this thesis was to compare the results of these two airway sampling methods in a population of 121 horses with signs related to respiratory disease and in 33 horses with no clinical respiratory signs. A secondary aim was to evaluate the cut-off values of TW and BALF cytology for neutrophils in order to justify their use in clinical practice. TW and BAL showed substantial agreement regarding neutrophils, and only 17.5% of the horses were classified differently (healthy vs. diseased), in contrast to some previous studies with higher disagreement. The neutrophil percentage was found to correlate between TW and BAL results. The sensitivity and specificity of TW were generally higher for TW than for BAL when estimated with conventional as well as with Bayesian statistical methods. The results of this study encourage the use of the current neutrophil cut-off values in clinical diagnosis of airway inflammation instead of elevating the cut-offs. In Study III, equine lower airway inflammation was investigated with regard to general anaesthesia. The complication rate in equine general anaesthesia is high relative to anaesthesia in several other species, postanaesthetic pneumonia being one of the potential complications. However, there is limited knowledge about the effects of general anaesthesia on inflammatory responses in the lungs and on systemic inflammatory markers such as serum amyloid A (SAA) in horses. For example, the level and duration of lower-airway neutrophilia that can be expected after anaesthesia in dorsal recumbency are unknown. Thus, our primary aim was to determine when lung inflammation reaches its maximum and how rapidly BALF cytology returns to baseline after anaesthesia in dorsal recumbency. A secondary aim was to investigate the possible effect of vatinoxan, a novel a2-adrenoceptor antagonist drug, on the BALF cytology results. Anaesthesia in dorsal recumbency resulted in no clinically relevant changes in airway cytology that could be differentiated from the effect of repeated BAL sampling. A marked increase in serum amyloid A was detected in some animals, while others showed no changes from baseline. Vatinoxan as premedication did not consistently affect lung cytology or blood inflammatory markers after anaesthesia. These combined studies on equine lower airway inflammation increase our understanding of mechanisms and factors related to pathophysiology and diagnostics of the condition in the horse. Lower airway inflammation is an important cause of impaired welfare and performance of horses. Broader understanding of the aspects related to the condition will aid in developing new improved clinical and diagnostic procedures and management strategies for equine inflammatory respiratory diseases. Alahengitysteiden steriilit tulehdussairaudet ovat yleisiä hevosilla rodusta, iästä ja hevosen käyttötarkoituksesta riippumatta. Hevosen kroonista alahengitysteiden tulehdusta kutsutaan astmaksi, sillä se muistuttaa monin tavoin ihmisen astmaa. Hevosen astma jaetaan oireilun voimakkuuden perusteella lievään/kohtalaiseen sekä vakavaan (kansankielisesti puhkuri). Tärkeimpänä astmalle altistavana tekijänä pidetään pitkäaikaista altistumista talli-ilman ja heinän pölylle ja homeitiöille. Astman oireita ovat mm. krooninen yskä ja sierainvuoto, huonontunut rasituksensietokyky sekä vakavassa muodossa myös hengitysvaikeus levossa (”astmakohtaus”). Aktiivisesta tutkimuksesta huolimatta hevosen astman patofysiologia ei ole vielä täysin tunnettu. Tämän väitöskirjan pääteemaa, hevosen alahengitysteiden tulehdusta, tutkittiin kolmesta eri näkökulmasta. Ensimmäisessä osatyössä selvitettiin extracellular matrix metalloproteinase inducer -proteiinin (EMMPRIN) patofysiologista merkitystä hevosen astmassa ja sen yhteyttä soluväliaineen gelatiinia hajottaviin matriksin metalloproteinaasientsyymeihin (MMP-2 ja MMP-9). Tutkimukseen osallistui 29 astmahevosta ja 15 oireetonta verrokkia. EMMPRIN-proteiinin todettiin olevan koholla vakavaa astmaa sairastavien hevosten keuhkohuuhtelunäytteissä, ja sen ilmentyminen korreloi MMP 2:n ja -9:n proteiini-ilmentymiseen sekä MMP-9:n aktiivisuuteen. Tässä tutkimuksessa selvitettiin ensimmäistä kertaa EMMPRIN:in suhdetta MMP-entsyymeihin kliinisessä astmassa. Toisessa osatyössä tutkittiin hevosen alahengitysteiden sairauksien diagnostiikassa käytettyjä näytteenottomenetelmiä. Tutkimuksessa verrattiin trakealima- ja keuhkohuuhtelunäytteen solukuvia toisiinsa sekä tarkasteltiin kliinisessä käytössä olevia solukuvaviitearvoja eri statistisin menetelmin 121:llä potilashevosella, joilla oli hengitystiesairauteen viittavia oireita, sekä 33 oireettomalla verrokilla. Menetelmien herkkyyttä ja tarkkuutta ei ole aikaisemmin arvioitu potilaiden diagnostiikassa. Tulosten perusteella trakealima- ja keuhkohuuhtelusolukuvat korreloivat hyvin neutrofiilisten solujen osalta, ja vain 17,5 % hevosista luokiteltiin näytteenottomenetelmien perusteella eri tavalla (terve vs. sairas), mikä on aiemmin raportoitua selvästi pienempi määrä. Trakealimanäytteen herkkyys ja tarkkuus olivat korkeammat keuhkohuuhteluun nähden. Viitearvojen tarkastelun perusteella nykyiset yleisimmin käytössä olevat viitearvot ovat edelleen käyttökelpoiset. Tutkimuksen tuloksia voidaan suoraan soveltaa kliinisessä käytössä hevosklinikoilla. Kolmannessa osatyössä selvitettiin yleisanestesian vaikutusta hevosen keuhkojen tulehdusvasteeseen ja toissijaisesti uuden lääkeaineen, vatinoksaanin, vaikutusta keuhkoihin. Hevosella ei tiedetä, aiheuttaako yleisanestesia selinmakuulla tulehdusvasteen keuhkoissa ja kauanko se kestää. Tutkimuksessa käytettiin kuutta koehevosta. Tulosten perusteella todettiin, että yleisanestesia selinmakuulla ei aiheuta merkittävää tulehdusvastetta hevosen keuhkoihin eikä vatinoksaanilla ole vaikutusta tulehdusvasteeseen. Joillain hevosilla kuitenkin veren akuutti tulehdusmarkkeri seerumin amyloidi A nousi selvästi anestesian jälkeen. Alahengitysteiden tulehdus on tärkeä hevosten hyvinvointia ja suorituskykyä huonontava ongelma. Tämän väitöskirjan tulokset lisäävät tietoa tulehduksen syntymekanismista sekä edistävät diagnostiikan kehittymistä.