Michela Bullone

Epidemiology of musculoskeletal injuries in a population of harness Standardbred racehorses in training

BackgroundThere is a substantial paucity of studies concerning musculoskeletal injuries in harness Standardbred racehorses. Specifically, little is known about the epidemiology of exercise-related musculoskeletal injuries. Most studies on this subject involve Thoroughbred racehorses, whose biomechanics and racing speed differ from Standardbred, making comparisons difficult. Here, a population of Standardbred racehorses trained at the same racecourse was studied over four years and a classification system for exercise-related musculoskeletal injuries was designed. The incidence rates of musculoskeletal injuries causing horses’ withdrawal from training for 15 days or longer were investigated. A mixed-effects Poisson regression model was used to estimate musculoskeletal injury rates and to describe significance of selected risk factors for exercise-related injuries in this population.ResultsA total of 356 trotter racehorses from 10 different stables contributed 8961 months at risk of musculoskeletal injuries. Four-hundred-and-twenty-nine injuries were reported and classified into 16 categories, based on their aetiology and anatomical localisation. The overall exercise-related injury rate was 4.79 per 100 horse months. When considering risk factors one by one in separate univariable analyses, we obtained the following results: rates did not differ significantly between genders and classes of age, whereas one driver seemed to cause fewer injuries than the others. Racing speed and racing intensity, as well as recent medical history, seemed to be significant risk factors (p < 0.001), while being shod or unshod during racing was not. On the other hand, when pooling several risk factors in a multivariable approach, only racing intensity turned out to be significant (p < 0.001), since racing speed and the racing intensity were partially confounded, being strongly correlated to one another.ConclusionCharacterizing epidemiology of exercise-related musculoskeletal injuries in trotter racehorses provides baseline incidence rate values. Incidence rates of stress fracture are lower in Standardbreds compared to Thoroughbreds, whereas the opposite is true for tendon and suspensory ligament injuries. In addition to identification of risk factors for musculoskeletal injuries among Standardbred racehorses, results suggest that racing intensity seems to be a protective predictor of risk and recent medical history could be used to identify horses at risk of injury.

PI3K and MAPKs regulate neutrophil migration toward the airways in heaves.

BACKGROUND Neutrophils accumulate in the airways of horses with heaves. They likely play an important role in the disease pathogenesis. Understanding the pathways regulating their migration may help identifying new therapeutic targets. HYPOTHESIS MAPK and PI3K pathways are involved in neutrophil migration toward the airway lumen in heaves. ANIMALS Twelve heaves-affected horses and 4 healthy horses. METHODS Migratory activity of bronchoalveolar lavage fluids (BALF) from horses with heaves and healthy horses was compared by means of a Boyden chamber. Involvement of MAPK and PI3K pathways in neutrophil migration was investigated by pretreating neutrophils with inhibitors of p38 MAPK, JNK, MEK1/2, and PI3K. The capacity of a p38 MAPK inhibitor at decreasing neutrophil chemotaxis toward the airways was also evaluated in vivo. RESULTS BALF from symptomatic heaves-affected horses induced a greater degree of chemokinesis (P = .0004) than BALF from healthy horses. Although all pathways tested were involved in neutrophil migration, inhibition of PI3K was most potent in vitro. An inhibitor of p38 MAPK administered before challenge in horses with heaves did not alter BALF chemokinetic properties. BALF neutrophil percentage and BALF migratory activity were positively correlated after 14 and 35 days of antigen challenge in healthy (P = .05; R(2) = 0.82) and heaves-affected horses (P = .03; R(2) = 0.76), respectively. CONCLUSIONS AND CLINICAL IMPORTANCE MAPK and PI3K pathways regulate neutrophil migration induced by BALF of horses with heaves. Inhibition of multiple pathways might be required to completely abolish BALF-induced neutrophil migratory activity and possibly inflammation in heaves.

Technical and physiological determinants of airway smooth muscle mass in endobronchial biopsy samples of asthmatic horses

Morphometric analyses of endobronchial biopsies are commonly performed in asthma research but little is known about the technical and physiological parameters contributing to measurement variability. We investigated factors potentially affecting biopsy size, quality, and airway smooth muscle (ASM) content in heaves, an asthma-like disease of horses. Horses with heaves in clinical exacerbation (n = 6) or remission (n = 6) from the disease and six controls were studied using a crossover design. The effect of disease status, age, bronchodilation, biopsy forceps type, and carina size on total biopsy area (Atot), ASM area (AASM), ASM% (AASM/Atot), and histologic quality were assessed. Concordance among different measuring techniques was also assessed. Compared with other groups, horses with heaves in exacerbation yielded larger biopsies (P < 0.05). Better quality biopsies were obtained from carinae of small size compared with large ones (P = 0.02), and carina size and forceps type significantly affected the ASM content of the biopsy (interaction, P < 0.05). AASM increased with age only in heaves-affected horses (r = 0.9, P < 0.05), and ASM% was negatively correlated with pulmonary resistance at 5 Hz in heaves-affected horses (r = -0.74, P = 0.01), likely because of the increased thickness of the extracellular matrix layer in this group (P = 0.01). In conclusion, disease status, carina thickness, and the forceps used may significantly affect biopsy size, quality, and ASM content. Endobronchial biopsies are not appropriate samples for ASM quantification in heaves, and studies measuring ASM mass should not be compared when measuring techniques differ.

Endobronchial Ultrasound Reliably Quantifies Airway Smooth Muscle Remodeling in an Equine Asthma Model

Endobronchial ultrasonography (EBUS) revealed differences in the thickness of the layer representing subepithelial tissues (L2) between human asthmatics and controls, but whether this measurement correlates with airway smooth muscle (ASM) remodeling in asthma is unknown. In this study, we sought to determine the ability of EBUS to predict histological ASM remodeling in normal and equine asthmatic airways. We studied 109 isolated bronchi from the lungs of 13 horses. They underwent EBUS examination using a 30 MHz radial probe before being processed for histology. ASM remodeling parameters were evaluated in EBUS images (L2 thickness, L2 area, L2 area/internal perimeter [Pi] and L2 area/Pi2) and histological cuts (ASM area/Pi2), and compared. EBUS was then performed ex vivo on the lungs of 4 horses with heaves, an asthma-like condition of horses, and 7 controls to determine whether central bronchial remodeling could be detected with this technique. An optimized approach was developed based on data variability within airways, subjects, and groups, and then validated in 7 horses (3 controls, 4 with heaves) that underwent EBUS in vivo. L2 area was significantly associated to ASM area in isolated lungs (p<0.0001), in the absence of significant bias related to the airway size. Bronchial size significantly affected EBUS ASM-related parameters, except for L2 area/Pi2. L2 area/Pi2 was increased in the airways of asthmatic horses compared to controls, both ex vivo and in vivo (p<0.05). Bronchial histology confirmed our findings (AASM/Pi2 was increased in asthmatic horses compared to controls, p<0.05). In both horses with heaves and controls, L2 was composed of ASM for the outer 75% of its thickness and by ECM for the remaining inner 25%. In conclusion, EBUS reliably allows assessment of asthma-associated ASM remodeling of central airways in a non-invasive way.

Fluticasone/salmeterol reduces remodelling and neutrophilic inflammation in severe equine asthma

Asthmatic airways are inflamed and undergo remodelling. Inhaled corticosteroids and long-acting β2-agonist combinations are more effective than inhaled corticosteroid monotherapy in controlling disease exacerbations, but their effect on airway remodelling and inflammation remains ill-defined. This study evaluates the contribution of inhaled fluticasone and salmeterol, alone or combined, to the reversal of bronchial remodelling and inflammation. Severely asthmatic horses (6 horses/group) were treated with fluticasone, salmeterol, fluticasone/salmeterol, or with antigen avoidance for 12 weeks. Lung function, central and peripheral airway remodelling, and bronchoalveolar inflammation were assessed. Fluticasone/salmeterol and fluticasone monotherapy decreased peripheral airway smooth muscle remodelling after 12 weeks (p = 0.007 and p = 0.02, respectively). On average, a 30% decrease was observed with both treatments. In central airways, fluticasone/salmeterol reversed extracellular matrix remodelling after 12 weeks, both within the lamina propria (decreased thickness, p = 0.005) and within the smooth muscle layer (p = 0.004). Only fluticasone/salmeterol decreased bronchoalveolar neutrophilia (p = 0.03) to the same extent as antigen avoidance already after 8 weeks. In conclusion, this study shows that fluticasone/salmeterol combination decreases extracellular matrix remodelling in central airways and intraluminal neutrophilia. Fluticasone/salmeterol and fluticasone monotherapy equally reverse peripheral airway smooth muscle remodelling.

Development of a Semiquantitative Histological Score for the Diagnosis of Heaves Using Endobronchial Biopsy Specimens in Horses.

Background Remodeling of the peripheral airways persists during the asymptomatic phase of heaves. Assessing the histology of large bronchi could facilitate the diagnosis of heaves during remission of the disease. Hypothesis Airway inflammation and remodeling in endobronchial biopsy (EBB) specimens differentiate horses with heaves from controls, independently of their clinical status (exacerbation or remission). Animals Fourteen healthy horses and 24 horses with heaves. Methods A 14‐point scoring system assessing central bronchial wall inflammation and remodeling was developed. The score was validated by 2 pathologists using specimens obtained from 18 horses (6 controls, 6 with heaves exacerbation, and 6 with heaves remission) in which lung function had been assessed with impulse oscillometry. Clinical and research application of the score was evaluated using biopsy specimens obtained from 20 additional horses (8 controls, 6 with heaves exacerbation, and 6 with heaves remission). Results The score was repeatable (interclass correlation coefficient = 69%). It differentiated horses with heaves in exacerbation (mean ± SD: 6.2 ± 2.2) from those in remission (4.0 ± 1.0) and controls (3.6 ± 1.7, P < 0.0001). The histological scores of horses with heaves correlated with the ratio of respiratory resistance (R) at 5 and 10 Hz (R 5 : R 10 ratio, r = 0.65, P = 0.03), a parameter assessing airway obstruction. Conclusions and Clinical Significance The proposed histological scoring system correlates with the degree of airway obstruction measured by impulse oscillometry. However, it does not discriminate horses with heaves in remission from controls. Evaluation of EBB specimens might be considered in future research and clinical studies of respiratory diseases in horses.

Identification of surgically-induced longitudinal lesions of the equine deep digital flexor tendon in the digital flexor tendon sheath using contrast-enhanced ultrasonography: an ex-vivo pilot study

BackgroundLongitudinal tears in the lateral aspect of the deep digital flexor tendon are the most common causes of pain localised to the equine digital flexor tendon sheath. However conventional ultrasonographic techniques provide limited information about acute lesions. Ultrasonographic contrast agents are newly developed materials that have contributed to advancement in human diagnostic imaging. They are currently approved for intravenous use in human and animal models. In this study we described intrathecal use in the horse. This study was undertaken to evaluate the reliability of standard and angle contrast-enhanced ultrasonography to detect and characterize surgically-induced longitudinal lesions in the deep digital flexor tendons.In this pilot study surgically-induced lesions were created in the lateral aspect of the deep digital flexor tendon within the digital flexor tendon sheath in 10 isolated equine limbs to generate a replicable model for naturally occurring lesions. Another 10 specimens were sham operated. All the limbs were examined ultrasonographically before and shortly after the intrasynovial injection of an ultrasound contrast agent containing stabilised microbubbles. The images were blindly evaluated to detect the ability to identify surgically-created lesions. The deep digital flexor tendons were dissected and a series of slices were obtained. The depth of longitudinal defects identified with contrast-enhanced ultrasound scans was compared to the real extent of the lesions measured in the corresponding gross tendon sections.ResultsContrast-enhanced ultrasonography with both angle and standard approach provided a significant higher proportion of correct diagnoses compared to standard and angle contrast ultrasonography (p < 0.01). Contrast-enhanced ultrasonography reliably estimated the depth of surgically-induced longitudinal lesions in the deep digital flexor tendons.ConclusionContrast-enhanced ultrasound of the digital flexor tendon sheath could be an effective tool to detect intrasynovial longitudinal tears of the deep digital flexor tendon, although an in vivo study is required to confirm these results for naturally occurring lesions.

The Contribution of Oxidative Stress and Inflamm-Aging in Human and Equine Asthma

Aging is associated with a dysregulation of the immune system, leading to a general pro-inflammatory state of the organism, a process that has been named inflamm-aging. Oxidative stress has an important role in aging and in the regulation of immune responses, probably playing a role in the development of age-related diseases. The respiratory system function physiologically declines with the advancement of age. In elderly asthmatic patients, this may contribute to disease expression. In this review, we will focus on age-related changes affecting the immune system and in respiratory structure and function that could contribute to asthma occurrence, and/or clinical presentation in the elderly. Also, naturally occurring equine asthma will be discussed as a possible model for studying the importance of oxidative stress and immun-aging/inflamm-aging in humans.

Endoscopic Evaluation of Angiogenesis in the Large Airways of Horses with Heaves Using Narrow Band Imaging.

Background Heaves is a severe debilitating condition of horses, characterized by lower airway inflammation and permanent structural changes of the bronchial wall. Chronic inflammation promotes the formation of new vessels, a phenomenon known as angiogenesis. Narrow band imaging (NBI) endoscopy is a noninvasive technique that enhances the visualization of submucosal vessels, and commonly is employed for the study of angiogenesis in human patients. Objectives Using NBI, we aimed to determine whether or not the central airways of horses with heaves undergo angiogenesis. Animals Horses with heaves during exacerbation of the disease (n = 5) and healthy controls (n = 6). Methods A library of NBI images was established from previously recorded videoendoscopies. Images were acquired by an operator blinded to horse ID. Images were obtained from 3 sites: 130 from the trachea (14 ± 9.3 [mean ± SD] images per horse with heaves and 10 ± 5.4 from controls; P = .45), 58 from the carina (5.4 ± 3.2 from horses with heaves and 5.2 ± 2.8 fromn controls; P > .99) and 167 from the intermediate bronchi (17.8 ± 6.7 from horses with heaves and 13 ± 5.6 from controls; P = .17). Using dedicated stereology software (NewCAST, Visiopharm; Denmark), the volume density of superficial and deep vessels was calculated blindly by point counting at each site for all horses. Results In the trachea, the volume density of superficial vessels was increased in horses with heaves compared to controls (P = .02). No difference was found between groups for the volume density of both superficial and deep vessels at the carina or intermediate bronchi. Conclusion and Clinical Relevance NBI imaging of the airways was easily performed in standing sedated horses.

Bronchoalveolar lavage fluid neutrophilia is associated with the severity of pulmonary lesions during equine asthma exacerbations

BACKGROUND The severe form of equine asthma is associated with pathological changes of the peripheral airways and pulmonary parenchyma that are only partly described. Also, the relationship between these structural alterations and the percentage of neutrophils found within the airway lumen, assessed by bronchoalveolar lavage fluid (BALF) cytology, remains ill-defined. OBJECTIVE To examine the histological lesions associated with equine asthma during disease exacerbation and remission, and their relationship with lung function and BALF neutrophilia. STUDY DESIGN Observational retrospective study. METHODS Peripheral lung tissues, BALF cytology and lung function data from 61 horses (22 controls, 24 asthma exacerbations and 15 asthma remission) were obtained from an equine pulmonary tissue bank. Two pathologists semi-quantitatively assessed histological features, including airway wall inflammation, interstitial fibrosis, mucus cell hyperplasia, mucostasis, peribronchiolar metaplasia, presence of granuloma and the overall severity of these lesions. RESULTS Mucostasis, mucus cell hyperplasia, peribronchiolar metaplasia and interstitial fibrosis were associated with disease exacerbation (P≤0.05), and these changes were all attenuated during remission. Airway wall inflammation was greater in horses with asthma in exacerbation compared with horses with asthma in remission and control horses (P≤0.05). Acute (neutrophilic) airway wall inflammation was more frequently detected in asthmatic cases compared with control horses (P<0.0001) and was associated with BALF neutrophilia >5% in control horses (P = 0.002). The degree of bronchiolar inflammation was higher in asthmatic horses in remission stabled and treated pharmacologically compared with those kept on pasture (P = 0.04). MAIN LIMITATIONS Samples obtained from a convenient cohort of horses were studied. CONCLUSIONS Severely asthmatic horses present parenchymal and peribronchial/peribronchiolar lesions possibly contributing to the obstructive nature of the disease.