Mathilde Leclere

Heaves, an asthma-like disease of horses

Animal models have been developed to investigate specific components of asthmatic airway inflammation, hyper‐responsiveness or remodelling. However, all of these aspects are rarely observed in the same animal. Heaves is a naturally occurring disease of horses that combines these features. It is characterized by stable dust‐induced inflammation, bronchospasm and remodelling. The evaluation of horses during well‐controlled natural antigen exposure and avoidance in experimental settings allows the study of disease mechanisms in the asymptomatic and symptomatic stages, an approach rarely feasible in humans. Also, the disease can be followed over several years to observe the cumulative effect of repeated episodes of clinical exacerbation or to evaluate long‐term treatment, contrasting most murine asthma models. This model has shown complex gene and environment interactions, the involvement of both innate and adaptive responses to inflammation, and the contribution of bronchospasm and tissue remodelling to airway obstruction, all occurring in a natural setting. Similarities with the human asthmatic airways are well described and the model is currently being used to evaluate airway remodelling and its reversibility in ways that are not possible in people for ethical reasons. Tools including antibodies, recombinant proteins or gene arrays, as well as methods for sampling tissues and assessing lung function in the horse are constantly evolving to facilitate the study of this animal model. Research perspectives that can be relevant to asthma include the role of neutrophils in airway inflammation and their response to corticosteroids, systemic response to pulmonary inflammation, and maintaining athletic capacities with early intervention.

Corticosteroids and Antigen Avoidance Decrease Airway Smooth Muscle Mass in an Equine Asthma Model

Recent studies suggest that airway smooth muscle remodeling is an early event in the course of asthma. Little is known of the effects of long-term antigen avoidance and inhaled corticosteroids on chronically established airway remodeling. We sought to measure the effects of inhaled corticosteroids and antigen avoidance on airway remodeling in the peripheral airways of horses with heaves, a naturally occurring asthma-like disease. Heaves-affected adult horses with ongoing airway inflammation and bronchoconstriction were treated with fluticasone propionate (with and without concurrent antigen avoidance) (n = 6) or with antigen avoidance alone (n = 5). Lung function and bronchoalveolar lavage were performed at multiple time points, and peripheral lung biopsies were collected before and after 6 and 12 months of treatment. Lung function improved more quickly with inhaled corticosteroids, but eventually normalized in both groups. Inflammation was better controlled with antigen avoidance. During the study period, corrected smooth muscle mass decreased from 12.1 ± 2.8 × 10(-3) and 11.3 ± 1.2 × 10(-3) to 8.3 ± 1.4 × 10(-3) and 7.9 ± 1.0 × 10(-3) in the antigen avoidance and fluticasone groups, respectively (P = 0.03). At 6 months, smooth muscle mass was significantly smaller compared with baseline only in the fluticasone-treated animals. The subepithelial collagen area was lower at 12 months than at baseline in both groups. During the study period, airway smooth muscle remodeling decreased by approximately 30% in both groups, although the decrease was faster in horses receiving inhaled corticosteroids. Inhaled corticosteroids may accelerate the reversal of smooth muscle remodeling, even if airway inflammation is better controlled with antigen avoidance.

Markers of Systemic Inflammation in Horses with Heaves

BACKGROUND Systemic inflammation in horses with heaves is poorly characterized. OBJECTIVES To assess acute phase proteins (APP) and inflammatory cytokine profiles in serum of healthy horses and horses with heaves. ANIMALS Six healthy horses and 6 heaves-affected horses belonging to the University of Montreal. METHODS Prospective, observational study. Healthy and heaves-affected control horses were exposed to a 30-day natural challenge with hay and straw to induce clinical exacerbation of heaves. Serum samples were obtained by venipuncture before (T0) as well as after 7 (T7) and 30 days (T30) of stabling. Serum APP (haptoglobin, serum amyloid A protein [SAA] and C-reactive protein [CRP]) and cytokines (IL-2, IL-4, IFN-α, IL-10, IFN-γ, and CCL-2) were measured using singleplex or multiplex ELISA. RESULTS Serum haptoglobin concentrations were significantly higher in heaves-affected horses at all time points with no overlap with those of healthy controls. They were also significantly increased by antigen challenge in both controls (T7) and horses with heaves (T7 and T30). Serum SAA was detected more frequently in heaves-affected horses compared with healthy controls at T7. There was no difference in serum concentrations of CRP, IL-10, IFN-γ, and CCL-2 between groups, whereas IL-2, IL-4, and IFN-α remained undetectable in all samples. CONCLUSIONS AND CLINICAL IMPORTANCE In heaves, haptoglobin is a marker of both acute and chronic systemic inflammation, whereas high concentrations of SAA indicate acute inflammation.

Effect of Antigenic Exposure on Airway Smooth Muscle Remodeling in an Equine Model of Chronic Asthma

Recent studies suggest that airway smooth muscle remodeling is an early event in asthma, but whether it remains a dynamic process late in the course of the disease is unknown. Moreover, little is known about the effects of an antigenic exposure on chronically established smooth muscle remodeling. We measured the effects of antigenic exposure on airway smooth muscle in the central and peripheral airways of horses with heaves, a naturally occurring airway disease that shares similarities with chronic asthma. Heaves-affected horses (n = 6) and age-matched control horses (n = 5) were kept on pasture before being exposed to indoor antigens for 30 days to induce airway inflammation and bronchoconstriction. Peripheral lung and endobronchial biopsies were collected before and after antigenic exposure by thoracoscopy and bronchoscopy, respectively. Immunohistochemistry and enzymatic labeling were used for morphometric analyses of airway smooth muscle mass and proliferative and apoptotic myocytes. In the peripheral airways, heaves-affected horses had twice as much smooth muscle as control horses. Remodeling was associated with smooth muscle hyperplasia and in situ proliferation, without reduced apoptosis. Further antigenic exposure had no effect on the morphometric data. In central airways, proliferating myocytes were increased compared with control horses only after antigenic exposure. Peripheral airway smooth muscle mass is stable in chronically affected animals subjected to antigenic exposure. This increased mass is maintained in a dynamic equilibrium by an elevated cellular turnover, suggesting that targeting smooth muscle proliferation could be effective at decreasing chronic remodeling.

Evaluation of a modified thrombelastography assay initiated with recombinant human tissue factor in clinically healthy horses

BACKGROUND Thrombelastography (TEG) is used to evaluate the viscoelastic properties of blood during clotting and provides a global assessment of hemostasis and clot lysis. TEG analysis initiated with recombinant human tissue factor (TF) has not been evaluated in clinically healthy horses. OBJECTIVES The purpose of this study was to determine whether TEG results are affected by the time elapsed between sampling and analysis (storage time) of equine blood samples and to establish a preliminary equine reference interval for a modified TEG assay, using recombinant human TF to initiate coagulation. METHODS Citrated blood samples were obtained from 20 clinically healthy adult horses. Thirteen samples were stored for 30, 60, and 120 minutes at room temperature before TEG analysis. Coagulation was initiated by adding 20 microL of CaCl(2) to 330 microL of blood and 10 microL of diluted recombinant TF for a final dilution of 1:3600. Reaction (R) and clotting (K) times, angle (alpha), and maximum amplitude (MA) were compared between time points. A preliminary reference interval (minimum-maximum values) was determined using data from all 20 horses after 30 minutes of sample storage. RESULTS There was a significant effect of storage time on R, K, and alpha but not MA. Reference intervals were: R, 3.65-6.4 minutes; K, 1.8-5.45 minutes; alpha, 33.4-66.2 degrees ; MA, 41.2-64.1 mm; lysis at 30 minutes post-MA (LY30), <2.75%; and lysis at 60 minutes post-MA (LY60), 1.55-9.5%. CONCLUSIONS TEG can be performed on equine citrated blood samples using recombinant human TF to activate clot formation. TEG parameters were significantly affected by storage time, suggesting an incomplete inhibition of coagulation in citrated blood.

Efficacy of oral prednisolone and dexamethasone in horses with recurrent airway obstruction in the presence of continuous antigen exposure.

REASONS FOR PERFORMING STUDY Orally administered prednisolone and dexamethasone are used commonly in the treatment of recurrent airway obstruction (RAO) in horses. However, the efficacy of prednisolone in improving pulmonary function during continuous antigen exposure has not been evaluated critically and there is little evidence supporting the efficacy of low-dose oral dexamethasone in the same conditions. HYPOTHESIS Oral prednisolone and dexamethasone improve pulmonary function in RAO under conditions of continuous antigen exposure, and dexamethasone is more effective than prednisolone at commonly used dosages. METHODS Using a randomised crossover design, prednisolone (2 mg/kg bwt) and dexamethasone (0.05 mg/kg bwt) were administered per os, s.i.d. for 7 days, to 7 horses during clinical exacerbation of the disease. Maximal difference in transpulmonary pressure (DeltaP(L)), lung resistance (R(L)) and elastance (E(L)) were measured before and after 3 and 7 days of treatment. RESULTS Prednisolone and dexamethasone improved pulmonary function significantly. However, the improvement was of greater magnitude after 3 and 7 days of treatment with dexamethasone compared to prednisolone. Also, after 7 days of treatment with dexamethasone, DeltaP(L) and R(L) were not different from values obtained when horses were on pasture, while all 3 pulmonary function parameters remained different from pasture values after prednisolone treatment. CONCLUSIONS Both corticosteroids improve pulmonary function, in spite of continuous antigen exposure. However, oral dexamethasone at 0.05 mg/kg bwt is more effective than prednisolone at 2 mg/kg bwt in the treatment of RAO. POTENTIAL RELEVANCE Prednisolone was shown, for the first time, to our knowledge, to improve the pulmonary function of horses with RAO in the presence of continuous antigen exposure. This study also demonstrates the efficacy of low-dose oral dexamethasone in reversing airway obstruction in these conditions.

Effect of long-term fluticasone treatment on immune function in horses with heaves.

BACKGROUND Corticosteroids currently are the most effective pharmacological treatment available to control heaves in horses. Systemically administered corticosteroids have been shown to alter immune response in horses, humans, and other species. Aerosolized administration theoretically minimizes systemic adverse effects, but the effect of inhaled corticosteroids on immune function has not been evaluated in horses. OBJECTIVES To evaluate the effects of prolonged administration of inhaled fluticasone on the immune system of heaves-affected horses. ANIMALS Heaves-affected horses were treated with inhaled fluticasone (n = 5) for 11 months or received environmental modifications only (n = 5). METHODS Prospective analysis. Clinical parameters and CBC, lymphocyte subpopulations and function, and circulating neutrophil gene expression were sequentially measured. Primary and anamnestic immune responses also were evaluated by measuring antigen-specific antibodies in response to vaccination with bovine viral antigen and tetanus toxoid, respectively. RESULTS No clinical adverse effects were observed and no differences in immune function were detected between treated and untreated horses. CONCLUSIONS AND CLINICAL IMPORTANCE The treatment of heaves-affected horses with inhaled fluticasone at therapeutic dosages for 11 months has no significant detectable effect on innate and adaptive (both humoral and cell-mediated) immune parameters studied. These results suggest that prolonged administration of fluticasone would not compromise the systemic immune response to pathogens nor vaccination in adult horses.

Profiling of differentially expressed genes using suppression subtractive hybridization in an equine model of chronic asthma.

Background Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma. Objective To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition. Methods Eleven adult horses (6 heaves-affected and 5 controls) were studied while horses with heaves were in clinical remission (Pasture), and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge). Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH), lung cDNAs of controls (Pasture and Challenge) and asymptomatic heaves-affected horses (Pasture) were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge). The differential expression of selected genes of interest was confirmed using quantitative PCR assay. Results Horses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways. Conclusions Pathways representing new possible targets for anti-inflammatory and anti-remodeling therapies for asthma were identified. The findings of genes previously associated with asthma validate this equine model for gene expression studies.

Evaluation of a thoracoscopic technique using ligating loops to obtain large lung biopsies in standing healthy and heaves-affected horses.

OBJECTIVE To evaluate use of pre-tied ligating loop to perform thoracoscopic, large lung biopsy in normal and heaves-affected horses. STUDY DESIGN Prospective clinical study. ANIMALS Normal (n=5) and heaves-affected (n=6) horses. METHODS Lung biopsies, 1 from each hemithorax, were collected thoracoscopically using a pre-tied ligating loop. Horses were either normal (C) or heaves-affected with the latter being in remission (Ha) for the initial biopsy and in exacerbation (Hs) for the 2nd biopsy. Clinical variables, PaO(2), and PaCO(2) were used to determine the effect of surgical biopsy. Postoperative pneumothorax was monitored by serial thoracic radiographic examinations. RESULTS Thoracoscopic lung biopsy (n=29, 22 procedures) was well tolerated by all horses. Complication rate was 31%, including 8 ligature slippage and 1 pulmonary hemorrhage. Intranasal oxygen was administered intraoperatively to 6 horses (2 C, 1 Ha, 3 Hs) with severe hypoxemia or labored breathing. There was a significant decrease in PaO(2) during surgery in horses not supplemented with oxygen. Postoperative pneumothorax (21/22 procedures) detected radiographically resolved within 3 weeks. CONCLUSION Thoracoscopic lung biopsy using pre-tied ligating loops was minimally invasive, relatively inexpensive, and fairly efficient. Heaves-affected horses tolerated the surgery well, even when in exacerbation; however, the technique was associated with non life-threatening complications in 31% of the biopsies, most of which required correction with additional ligating loops or more sophisticated instrumentation. CLINICAL RELEVANCE Using laparoscopic pre-tied ligating loop for thoracoscopically-assisted lung biopsy can be considered in horses with normal and impaired lung function but alternative instrumentation and access to intranasal oxygen must be available to the surgeon in case of complications.

Thoracoscopic lung biopsies in heaves-affected horses using a bipolar tissue sealing system.

Objective: To validate the use of the LigaSure™ Vessel Sealing System (LVSS) to perform thoracoscopic lung tissue biopsies in heaves-affected horses. Study design: Prospective clinical study. Animals: Heaves-affected horses (n=12). Methods: Lung biopsies (n=34) were collected with the LVSS (2–4 biopsies/horse) in horses with and without clinical signs of heaves. Thoracoscope (13th intercostal space [ICS]) and 2 instruments (between the 12–15th ICS) portals were used. Selected clinical and arterial blood gas variables were monitored. Postoperative pneumothorax was evaluated. Depth of thermal injury to the surrounding tissue and representativeness of the biopsies were determined. Results: Mean surgical time was 22.9±8.0 minutes. The complication rate was 5.6%, and primarily related to a focal inadequate sealing of the biopsy margin. Five horses in exacerbation required intraoperative intranasal O2. Mean PaO2 was significantly lower in heaves-affected horses with clinical signs compared with those without clinical signs. Postoperative pneumothorax was detected radiographically after 20 of the 34 procedures. One horse with clinical signs of heaves developed a fatal tension pneumothorax 5 days postoperatively despite close radiographic monitoring. Conclusion: Thoracoscopic lung biopsy using LVSS is a rapid and effective technique to harvest peripheral lung tissues from heaves-affected horses. Although the complication rate was tolerable, tension pneumothorax was a potential life-threatening complication because of incomplete lung sealing. Clinical Relevance: LVSS can be used with relative safety to perform thoracoscopic lung biopsy, but close postoperative monitoring is necessary to avoid tension pneumothorax.OBJECTIVE To validate the use of the LigaSure™ Vessel Sealing System (LVSS) to perform thoracoscopic lung tissue biopsies in heaves-affected horses. STUDY DESIGN Prospective clinical study. ANIMALS Heaves-affected horses (n=12). METHODS Lung biopsies (n=34) were collected with the LVSS (2-4 biopsies/horse) in horses with and without clinical signs of heaves. Thoracoscope (13th intercostal space [ICS]) and 2 instruments (between the 12-15th ICS) portals were used. Selected clinical and arterial blood gas variables were monitored. Postoperative pneumothorax was evaluated. Depth of thermal injury to the surrounding tissue and representativeness of the biopsies were determined. RESULTS Mean surgical time was 22.9±8.0 minutes. The complication rate was 5.6%, and primarily related to a focal inadequate sealing of the biopsy margin. Five horses in exacerbation required intraoperative intranasal O(2) . Mean PaO(2) was significantly lower in heaves-affected horses with clinical signs compared with those without clinical signs. Postoperative pneumothorax was detected radiographically after 20 of the 34 procedures. One horse with clinical signs of heaves developed a fatal tension pneumothorax 5 days postoperatively despite close radiographic monitoring. CONCLUSION Thoracoscopic lung biopsy using LVSS is a rapid and effective technique to harvest peripheral lung tissues from heaves-affected horses. Although the complication rate was tolerable, tension pneumothorax was a potential life-threatening complication because of incomplete lung sealing. CLINICAL RELEVANCE LVSS can be used with relative safety to perform thoracoscopic lung biopsy, but close postoperative monitoring is necessary to avoid tension pneumothorax.