Fetlock Joint Angle Pattern and Range of Motion Quantification Using Two Synchronized Wearable Inertial Sensors per Limb in Sound Horses and Horses with Single Limb Naturally Occurring Lameness.

Fetlock joint angle (FJA) pattern is a sensitive indicator of lameness. The first aim of this study is to describe a network of inertial measurement units system (IMUs) for quantifying FJA simultaneously in all limbs. The second aim is to evaluate the accuracy of IMUs for quantifying the sagittal plane FJA overground in comparison to bi-dimensional (2-D) optical motion capture (OMC). 14 horses (7 free from lameness and 7 lame) were enrolled and analyzed with both systems at walk and trot on a firm surface. All enrolled horses were instrumented with 8 IMUs (a pair for each limb) positioned at the dorsal aspect of the metacarpal/metatarsal bone and pastern and acquiring data at 200 Hz. Passive markers were glued on the center of rotation of carpus/tarsus, fetlock, and distal interphalangeal joint, and video footages were captured at 60 Hz and digitalized for OMC acquisition. The IMU system accuracy was reported as Root Mean Square Error (RMSE) and Pearson Correlation Coefficient (PCC). The Granger Causality Test (GCT) and the Bland−Altman analysis were computed between the IMUs and OMC patterns to determine the agreement between the two systems. The proposed IMU system was able to provide FJAs in all limbs using a patented method for sensor calibration and related algorithms. Fetlock joint range of motion (FJROM) variability of three consecutive strides was analyzed in the population through 3-way ANOVA. FJA patterns quantified by IMUs demonstrated high accuracy at the walk (RMSE 8.23° ± 3.74°; PCC 0.95 ± 0.03) and trot (RMSE 9.44° ± 3.96°; PCC 0.96 ± 0.02) on both sound (RMSE 7.91° ± 3.19°; PCC 0.97 ± 0.03) and lame horses (RMSE 9.78° ± 4.33°; PCC 0.95 ± 0.03). The two systems' measurements agreed (mean bias around 0) and produced patterns that were in temporal agreement in 97.33% of the cases (p < 0.01). The main source of variability between left and right FJROM in the population was the presence of lameness (p < 0.0001) and accounted for 28.46% of this total variation. IMUs system accurately quantified sagittal plane FJA at walk and trot in both sound and lame horses.

Giant Multinucleated Cells Are Associated with Mastocytic Inflammatory Signature Equine Asthma.

Equine asthma is currently diagnosed by the presence of increased neutrophil (>5%), mast cell (>2%), and/or eosinophil (>1%) differential cell count. Macrophages are normal resident cells within the alveoli. Their presence in BALF is considered normal, but the clinical implication of the presence of activated or fused macrophages (giant multinucleated cells, GMC) is currently overlooked. We aimed to assess the prevalence, cytological determinants, and clinical significance of increased GMC counts in BALF of 34 asthmatic horses compared to 10 controls. Counts were performed on 15 randomly selected high magnification fields per cytospin slide (40×), and expressed as GMC:single macrophage (GMC:M) ratio. Regression models were used for statistical analysis. GMC was frequently observed in both asthmatic and control horses, with an increased prevalence of equine asthma (p = 0.01). GMC:M ratio was significantly higher in severe vs. mild to moderate equine asthmatic and control horses. In asthmatic horses, an increased GMC:M ratio was significantly associated with BALF mastocytosis (p = 0.01), once adjusting for age and the presence and severity of clinical signs of the horses. Tachypnea was the only clinical sign that tended to be positively associated with GMC:M ratio after adjustment (p = 0.08). In conclusion, our data suggest that a relationship might exist between molecular mechanisms regulating GMC formation and mast cell recruitment in the equine lung. The same mechanisms could lead to tachypnea even in the absence of respiratory effort at rest. We suggest including GMC count in the basic cytological assessment of BALF samples to gain more insights into their role in equine asthma.

Effect of intravenous tiludronate disodium administration on the radiographic progression of osteoarthritis of the fetlock joint in Standardbred racehorses.

OBJECTIVE: To compare the effects of tiludronate disodium and 3 other medical treatments on clinical and radiographic findings and biomarkers of disease progression in horses with osteoarthritis of the fetlock joint. ANIMALS: 100 Standardbred racehorses with spontaneous traumatic injury of the fetlock joint. PROCEDURES: Horses were retrospectively grouped by whether they received tiludronate IV or triamcinolone acetonide and hyaluronan, polysulfated glycosaminoglycan, or interleukin-1 receptor antagonist protein intra-articularly. Data were collected on clinical, radiographic, and ultrasonographic findings and results for serum and synovial samples obtained before and 6 months after treatment. Lameness score, joint flexion test response, radiographic score, serum concentrations of tumor necrosis factor-α and carboxy-terminal telopeptides of collagen types I and II (CTX-I and II, respectively), and synovial fluid concentrations of interleukin-1ß, prostaglandin E2, and CTX-II were compared among treatments. RESULTS: All treatments resulted in a significant improvement in lameness score and joint flexion test response at 6 months. In horses that received triamcinolone acetonide and hyaluronan, synovial fluid interleukin-1ß, prostaglandin E2, and CTX-II concentrations decreased after treatment, suggesting this treatment inhibited progression of hyaline cartilage degeneration and inflammatory processes. Horses that received tiludronate were the only group that had a decrease in radiographic score and serum CTX-I concentration after treatment, supporting the effect of tiludronate on bone metabolism. Tiludronate treatment was also followed by increases in serum and synovial fluid concentrations of CTX-II, a marker of cartilage damage. CONCLUSIONS AND CLINICAL RELEVANCE: Tiludronate appeared to inhibit the radiographic progression of osteoarthritis in high-motion joints of racehorses at 6 months after treatment by inhibiting subchondral bone remodeling. Whether this effect was associated with a worsening of progressive cartilage damage remains to be ascertained.

Radiofrequency Thermoablation On Ex Vivo Animal Tissues: Changes on Isolated Swine Thyroids.

The use of Radiofrequency thermoablation (RFA) for treating large thyroid nodules is limited by the modest efficiency of the available systems in terms of volume of the ablation zones (AZs). This increases the risk of incomplete ablation of the nodule. Systems employing perfused electrodes have been developed to increase the volume of the AZ. Aim of this study is to compare the size of the AZ induced by RFA systems using internally cooled perfused vs. non-perfused electrodes in swine thyroids. RFAs were performed on 40 freshly isolated swine thyroids using both systems. The perfused system was tested using 0.9% saline, 7% and 18% hypertonic saline solutions. Energy delivery to the tissue was stopped when tissue conductivity dropped (real life simulations) and after an established time of 20 seconds (controlled duration). Following RFA, thyroids were transversally and longitudinally cut. Photographs were taken for macroscopic morphometry of the ablated zones before and after formalin fixation, to evaluate tissue shrinkage. Microscopic morphometry was performed on PAS stained sections. In real life simulation experiments, gross morphometry revealed that AZs produced with electrodes perfused using 7.0% saline are larger compared to isotonic saline. Microscopically, all the conditions tested using the perfused system produced larger AZs compared to non-perfused system after 20 seconds of RFA. In conclusion, the perfusion with 7.0% NaCl solution increased the electrical conductivity of the tissue in real life simulations, resulting in larger ablated areas compared to the use of isotonic saline.

Within-Breath Oscillatory Mechanics in Horses Affected by Severe Equine Asthma in Exacerbation and in Remission of the Disease.

Oscillometry is a technique that measures the resistance (R) and the reactance (X) of the respiratory system. In humans, analysis of inspiratory and expiratory R and X allows to identify the presence of tidal expiratory flow limitation (EFLt). The aim of this study was to describe inspiratory and expiratory R and X measured by impulse oscillometry system (IOS) in horses with severe asthma (SEA) when in clinical remission (n = 7) or in exacerbation (n = 7) of the condition. Seven healthy, age-matched control horses were also studied. Data at 3, 5, and 7 Hz with coherence > 0.85 at 3 Hz and >0.9 at 5 and 7 Hz were considered. The mean, inspiratory and expiratory R and X and the difference between inspiratory and expiratory X (ΔX) were calculated at each frequency. The data from the three groups were statistically compared. Results indicated that in horses during exacerbation of severe asthma, X during expiratory phase is more negative than during inspiration, such as in humans in presence of EFLt. The evaluation of X during inspiration is promising in discriminating between horses with SEA in remission and control horses.

Severe asthma is associated with a remodeling of the pulmonary arteries in horses.

Pulmonary hypertension and cor pulmonale are complications of severe equine asthma, as a consequence of pulmonary hypoxic vasoconstriction. However, as pulmonary hypertension is only partially reversible by oxygen administration, other etiological factors are likely involved. In human chronic obstructive pulmonary disease, pulmonary artery remodeling contributes to the development of pulmonary hypertension. In rodent models, pulmonary vascular remodeling is present as a consequence of allergic airway inflammation. The present study investigated the presence of remodeling of the pulmonary arteries in severe equine asthma, its distribution throughout the lungs, and its reversibility following long-term antigen avoidance strategies and inhaled corticosteroid administration. Using histomorphometry, the total wall area of pulmonary arteries from different regions of the lungs of asthmatic horses and controls was measured. The smooth muscle mass of pulmonary arteries was also estimated on lung sections stained for α-smooth muscle actin. Reversibility of vascular changes in asthmatic horses was assessed after 1 year of antigen avoidance alone or treatment with inhaled fluticasone. Pulmonary arteries showed increased wall area in apical and caudodorsal lung regions of asthmatic horses in both exacerbation and remission. The pulmonary arteries smooth muscle mass was similarly increased. Both treatments reversed the increase in wall area. However, a trend for normalization of the vascular smooth muscle mass was observed only after treatment with antigen avoidance, but not with fluticasone. In conclusion, severe equine asthma is associated with remodeling of the pulmonary arteries consisting in an increased smooth muscle mass. The resulting narrowing of the artery lumen could enhance hypoxic vasoconstriction, contributing to pulmonary hypertension. In our study population, the antigen avoidance strategy appeared more promising than inhaled corticosteroids in controlling vascular remodeling. However, further studies are needed to support the reversibility of vascular smooth muscle mass remodeling after asthma treatment.

Equine Asthma: Current Understanding and Future Directions.

The 2019 Havemeyer Workshop brought together researchers and clinicians to discuss the latest information on Equine Asthma and provide future research directions. Current clinical and molecular asthma phenotypes and endotypes in humans were discussed and compared to asthma phenotypes in horses. The role of infectious and non-infectious causes of equine asthma, genetic factors and proposed disease pathophysiology were reviewed. Diagnostic limitations were evident by the limited number of tests and biomarkers available to field practitioners. The participants emphasized the need for more accessible, standardized diagnostics that would help identify specific phenotypes and endotypes in order to create more targeted treatments or management strategies. One important outcome of the workshop was the creation of the Equine Asthma Group that will facilitate communication between veterinary practice and research communities through published and easily accessible guidelines and foster research collaboration.

Characterization of the ablation zones produced by three commercially available systems from a single vendor for radiofrequency thermoablation in an ex vivo swine liver model.

BACKGROUND: Radiofrequency Ablation (RFA) is rarely performed in veterinary medicine. A rationale exists for its use in selected cases of canine liver tumours. RFA induces ablation zones of variable size and geometry depending on the technique used and on the impedance of the targeted organ. OBJECTIVES: (a) to describe the geometry and reproducibility of the ablation zones produced by three commercially available systems from a single company, using isolated swine liver parenchyma as a model for future veterinary applications in vivo; (b) to study the effects of local saline perfusion into the ablated parenchyma through the electrode tip and of single versus double passage of the electrode on size, geometry and reproducibility of the ablation zones produced. METHODS: Size, and geometry of ablation zones reproduced in six livers with one cooled and perfused (saline) and two cooled and non-perfused systems, after single or double passage (n = 6/condition), were assessed macroscopically on digitalized images by a blinded operator. Longitudinal and transverse diameters, equivalent diameter, estimated volume and roundness index were measured. Reproducibility was assessed as coefficient of variation. RESULTS AND CONCLUSIONS: Ablation zone reproducibility was higher when expressed in terms of ablation zone diameters than estimated volume. Local saline perfusion of the parenchyma through the electrode tip during RFA increased the ablation zone longitudinal diameter. Ablation zone estimated volume increased with saline perfusion only when double passage was performed. These data may provide useful information for those clinicians who intend to include RFA as an additive tool in veterinary interventional radiology.

Use of a biopolymer delivery system to investigate the influence of interleukin-4 on recruitment of neutrophils in equids.

OBJECTIVE: To use a biopolymer delivery system to investigate the ability of interleukin (IL)-4 to recruit neutrophils into subcutaneous tissues of equids. ANIMALS: 16 horses and 2 ponies. PROCEDURES: Animals were assigned to 3 experiments (6/experiment). Effects of recombinant equine (Req) IL-4 (100, 250, or 500 ng/site) versus a positive control (ReqIL-8; 100 ng, 250 ng, or 1 µg/site) and a negative control (Dulbecco PBSS or culture medium) on neutrophil chemotaxis were assessed after SC injection into the neck with an injectable biopolymer used as the vehicle. Tissue samples including the biopolymer plug were collected by biopsy at various time points from 3 hours to 7 days after injection. Neutrophil infiltration was evaluated by histologic scoring (experiments 1, 2, and 3) or flow cytometry (experiment 3). RESULTS: Histologic neutrophil infiltration scores did not differ significantly among treatments at most evaluated time points. On flow cytometric analysis, log-transformed neutrophil counts in biopsy specimens were significantly greater for the ReqIL-8 treatment (1 µg/site) than the negative control treatment at 3 but not 6 hours after injection; results did not differ between ReqIL-4 and control treatments at either time point. Negative control treatments induced an inflammatory response in most equids in all experiments. CONCLUSIONS AND CLINICAL RELEVANCE: Flow cytometry was a more reliable method to estimate neutrophil migration than histologic score analysis. The ReqIL-4 treatment did not induce a detectable neutrophil response, compared with the negative control treatment in this study. Evidence of inflammation in negative control samples suggested the biopolymer is not a suitable vehicle for use in equids.

The equine asthma model of airway remodeling: from a veterinary to a human perspective.

Human asthma is a complex and heterogeneous disorder characterized by chronic inflammation, bronchospasm and airway remodeling. The latter is a major determinant of the structure-function relationship of the respiratory system and likely contributes to the progressive and accelerated decline in lung function observed in patients over time. Corticosteroids are the cornerstone of asthma treatment. While their action on inflammation and lung function is well characterized, their effect on remodeling remains largely unknown. An important hindrance to the study of airway remodeling as a major focus in asthma research is the lack of reliable non-invasive biomarkers. In consequence, the physiologic and clinical consequences of airway wall thickening and altered composition are not well understood. In this perspective, equine asthma provides a unique and ethical (non-terminal) preclinical model for hypothesis testing and generation. Severe equine asthma is a spontaneous disease affecting adult horses characterized by recurrent and reversible episodes of disease exacerbations. It is associated with bronchoalveolar neutrophilic inflammation, bronchospasm, and excessive mucus secretion. Severe equine asthma is also characterized by bronchial remodeling, which is only partially improved by prolonged period of disease remission induced by therapy or antigen avoidance strategies. This review will focus on the similarities and differences of airway remodeling in equine and human asthma, on the strengths and limitations of the equine model, and on the challenges the model has to face to keep up with human asthma research.

Elevated serum IgE, oral corticosteroid dependence and IL-17/22 expression in highly neutrophilic asthma.

Information on the clinical traits associated with bronchial neutrophilia in asthma is scant, preventing its recognition and adequate treatment. We aimed to assess the clinical, functional and biological features of neutrophilic asthma and identify possible predictors of bronchial neutrophilia.The inflammatory phenotype of 70 mild-to-severe asthma patients was studied cross-sectionally based on the eosinophilic/neutrophilic counts in their bronchial lamina propria. Patients were classified as neutrophilic or non-neutrophilic. Neutrophilic asthma patients (neutrophil count cut-off: 47.17 neutrophils·mm-2; range: 47.17-198.11 neutrophils·mm-2; median: 94.34 neutrophils·mm-2) were further classified as high (≥94.34 neutrophils·mm-2) or intermediate (47.17- <94.34 neutrophils·mm-2). The effect of smoking ≥10 pack-years was also assessed.Neutrophilic asthma patients (n=38; 36 mixed eosinophilic/neutrophilic) had greater disease severity, functional residual capacity, inhaled corticosteroid (ICS) dose and exacerbations, and lower forced vital capacity (FVC) % pred and forced expiratory volume in 1 s (FEV1) reversibility than non-neutrophilic asthma patients (n=32; 28 eosinophilic and four paucigranulocytic). Neutrophilic asthma patients had similar eosinophil counts, increased bronchial CD8+, interleukin (IL)-17-F+ and IL-22+ cells, and decreased mast cells compared with non-neutrophilic asthma patients. FEV1 and FVC reversibility were independent predictors of bronchial neutrophilia in our cohort. High neutrophilic patients (n=21) had increased serum IgE levels, sensitivity to perennial allergens, exacerbation rate, oral corticosteroid dependence, and CD4+ and IL-17F+ cells in their bronchial mucosa. Excluding smokers revealed increased IL-17A+ and IL-22+ cells in highly neutrophilic patients.We provide new evidence linking the presence of high bronchial neutrophilia in asthma to an adaptive immune response associated with allergy (IgE) and IL-17/22 cytokine expression. High bronchial neutrophilia may discriminate a new endotype of asthma. Further research is warranted on the relationship between bronchoreversibility and bronchial neutrophilia.

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.

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

Asthmatic airways are inflamed and undergo remodelling. Inhaled corticosteroids and long-acting ß2-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.

Assessment of Airway Distensibility by the Forced Oscillation Technique: Reproducible and Potentially Simplifiable.

A non-invasive index of airway distensibility is required to track airway remodeling over time. The forced oscillation technique (FOT) provides such an index by measuring the change in respiratory system conductance at 5 Hz over the corresponding change in lung volume (ΔGrs5/ΔVL). To become useful clinically, this method has to be reproducible and easy to perform. The series of breathing maneuvers required to measure distensibility would be greatly facilitated if the difficulty of breathing below functional residual capacity (FRC) could be precluded and the number of maneuvers could be reduced. The distensibility at lung volumes below FRC is also reduced by several confounders, suggesting that excluding data points below FRC should provide a better surrogate for airway remodeling. The objectives of this study were to investigate the reproducibility of airway distensibility measured by FOT and to assess whether the method could be simplified to increase feasibility. Distensibility was measured at three separate occasions in 13 healthy volunteers. At each visit, three deflationary maneuvers were performed, each consisting of tidal breathing from total lung capacity (TLC) to residual volume by slowly decreasing the end-expiratory volume on each subsequent breath. Distensibility was calculated by using either all data points from TLC to residual volume (RV) or only data points from TLC to FRC for either all three or only the first two deflationary maneuvers. Intra-class correlation coefficients (ICC) were used to assess reproducibility and Bland-Altman analyses were used to assess the level of agreement between the differently calculated values of distensibility. The results indicate that distensibility calculated using all data points is reproducible (ICC = 0.64). Using data points from TLC to FRC slightly improved reproducibility (ICC = 0.68) and increased distensibility by 19.4%, which was expected as distensibility above FRC should not be affected by confounders. Using only data points within the first two maneuvers did not affect reproducibility when tested between TLC and FRC (ICC = 0.66). We conclude that a valuable measure of airway distensibility could potentially be obtained with only two deflationary maneuvers that do not require breathing below FRC. This simplified method would increase feasibility without compromising reproducibility.

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.

Asthma "of horses and men"--how can equine heaves help us better understand human asthma immunopathology and its functional consequences?

Animal models have been studied to unravel etiological, immunopathological, and genetic attributes leading to asthma. However, while experiments in which the disease is artificially induced have helped discovering biological and molecular pathways leading to allergic airway inflammation, their contribution to the understanding of the causality of the disease has been more limited. Horses naturally suffer from an asthma-like condition called "heaves" which presents sticking similarities with human asthma. It is characterized by reversible airway obstruction, airway neutrophilic inflammation, and a predominant Th2 immune response. This model allows one to investigate the role of neutrophils in asthma, which remains contentious, the regulation of chronic neutrophilic inflammation, and their possible implication in pulmonary allergic responses. Furthermore, the pulmonary remodeling features in heaves closely resemble those of human asthma, which makes this model unique to investigate the kinetics, reversibility, as well as the physiological consequences of tissue remodeling. In conclusion, heaves and asthma share common clinical presentation and also important immunological and tissue remodeling features. This makes heaves an ideal model for the discovery of novel pathways implicated in the asthmatic inflammation and associated tissue remodeling.