Analysis of genomic copy number variation in equine recurrent airway obstruction (heaves).

We explored the involvement of genomic copy number variants (CNVs) in susceptibility to recurrent airway obstruction (RAO), or heaves-an asthmalike inflammatory disease in horses. Analysis of 16 RAO-susceptible (cases) and six RAO-resistant (control) horses on a custom-made whole-genome 400K equine tiling array identified 245 CNV regions (CNVRs), 197 previously known and 48 new, distributed on all horse autosomes and the X chromosome. Among the new CNVRs, 30 were exclusively found in RAO cases and were further analyzed by quantitative PCR, including additional cases and controls. Suggestive association (P = 0.03; corrected P = 0.06) was found between RAO and a loss on chromosome 5 involving NME7, a gene necessary for ciliary functions in lungs and involved in primary ciliary dyskinesia in humans. The CNVR could be a potential marker for RAO susceptibility but needs further study in additional RAO cohorts. Other CNVRs were not associated with RAO, although several involved genes of interest, such as SPI2/SERPINA1 from the serpin gene family, which are associated with chronic obstructive pulmonary disease and asthma in humans. The SPI2/SERPINA1 CNVR showed striking variation among horses, but it was not significantly different between RAO cases and controls. The findings provide baseline information on the relationship between CNVs and RAO susceptibility. Discovery of new CNVs and the use of a larger population of RAO-affected and control horses are needed to shed more light on their significance in modulating this complex and heterogeneous disease.

Inflammatory stimuli differentially modulate the transcription of paracrine signaling molecules of equine bone marrow multipotent mesenchymal stromal cells.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease of joint tissues that causes articular cartilage erosion, osteophytosis and loss of function due to pain. Inflammation and inflammatory cytokines in synovial fluid (SF) contribute to OA progression. Intra-articular (IA) injections of multipotent mesenchymal stromal cells (MSCs) are employed to treat OA in both humans and animals. MSCs secrete paracrine pro-inflammatory and anabolic signaling molecules that promote tissue repair. The objective of this study was to investigate the effects of OASF on the gene expression of paracrine signaling molecules by MSCs. METHODS: The effects of Lipopolysaccharide (LPS) and interleukin (IL)-1ß as well as both normal (N) and osteoarthritis (OA) SF stimulations on the expression of paracrine pro-inflammatory (tumor necrosis factor (TNF)-α, IL-1ß, IL-8), modulatory (IL-6) and anabolic (vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-ß1 and insulin-like growth factor (IGF)-1) signaling molecules by equine bone marrow multipotent mesenchymal stromal cells (eBM-MSCs) was investigated employing reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In contrast with NSF, OASF significantly up-regulated the expression of VEGF in eBM-MSCs. Both NSF and OASF significantly down-regulated the expression of IL-1ß. LPS and IL-1ß significantly increased the expression of pro-inflammatory cytokines (TNF-α, IL-8 and IL-6; and IL-1ß and IL-8 respectively). DISCUSSION: We conclude that the transcription of paracrine signaling molecules in eBM-MSCs is modulated by SF. Furthermore, OA alters the properties of SF and the response of eBM-MSCs. Finally, the effects of LPS or IL-1ß stimulation are distinct to that observed following stimulations with OASF.

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.

Isolation of equine bone marrow-derived mesenchymal stem cells: a comparison between three protocols.

REASON FOR PERFORMING THE STUDY: There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. OBJECTIVE: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). MATERIALS AND METHODS: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteristics (self-renewal (CFU) and multilineage differentiation capacity) were determined for all 3 protocols. RESULTS: The mean +/- s.d. MSC yield from the Percoll protocol was significantly higher (6.8 +/- 3.8%) than the Classic protocol (1.3 +/- 0.7%). The numbers of MSCs recovered after 14 days culture per 10 ml BM sample were 24.0 +/- 12.1, 14.6 +/- 9.5 and 4.1 +/- 2.5 x 10(6) for the Percoll, Ficoll and Classic protocols, respectively, significantly higher for the Percoll compared with the Classic protocol. Importantly, no significant difference in cell viability or in osteogenic or chondrogenic differentiation was identified between the protocols. At Passage 0, cells retrieved with the Ficoll protocol had lower self-renewal capacity when compared with the Classic protocol but there was no significant difference between protocols at Passage 1. There were no significant differences between the 3 protocols for the global frequencies of CFUs at Passage 0 or 1. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that the Percoll gradient density separation protocol was the best in terms of MSC yield and self-renewal potential of the MSCs retrieved and that MSCs retrieved with the Ficoll protocol had the lowest self-renewal but only at passage 0. Then, the 3 protocols were equivalent. However, the Percoll protocol should be considered for equine MSC isolation to minimise culture time.

Intra-articular triamcinolone acetonide improves lung function in horses with severe asthma.

BACKGROUND: Locally administered corticosteroids are commonly used to treat joint diseases in sport and racehorses. As they are also the most potent drugs for the treatment of equine asthma, we hypothesised that the intra-articular corticosteroids used to treat joint diseases also improve the lung function in horses with severe asthma, thus potentially delaying the diagnosis of this common lung condition. OBJECTIVES: To compare the effects of intra-articular (IA) and intramuscular (IM) triamcinolone acetonide (TA) on lung function in horses with severe asthma. STUDY DESIGN: Randomised and controlled experiment on asthma-prone research animals. METHODS: Horses with severe asthma in clinical exacerbation were given either 20 mg of TA in both tarsocrural joints (n = 5; 40 mg/horse) or 40 mg of TA intramuscularly (n = 5). Lung function and TA serum concentrations were measured weekly for 35 days. TA serum concentrations were also evaluated on day 3. RESULTS: The pulmonary resistance (RL ) and elastance (EL ) values decreased by day 7 in the IA group (P<0.0001 and P = 0.003, respectively) and by day 14 in the IM group (P = 0.002 and 0.03, respectively). Lung function was improved up to days 21 and 28 in the IA and IM groups, respectively, when compared with baseline. TA serum levels were below the quantification limit (100 pg/ml) for 4 and 3 of the 5 horses in the IA and IM groups, respectively, on day 7. The area under the curve for RL , EL and the serum concentrations of TA were similar in both groups. MAIN LIMITATIONS: The response of horses with severe asthma might differ from that of high-performance horses with mild/moderate asthma. CONCLUSIONS: Intra-articular administration of TA improves lung function in horses with severe asthma, an effect that persists when TA serum concentration is below the quantification level that is employed as a threshold by the International Association of Racing Commissioners.

Genomic and non-genomic effects of dexamethasone on equine peripheral blood neutrophils.

BACKGROUND: Glucocorticoids have potent anti-inflammatory properties and are frequently used for the treatment of domestic animal species, including horses. They induce a down-regulation of multiple inflammatory pathways through both genomic and non-genomic effects. Currently, little is known on the effects of glucocorticoids on equine peripheral blood neutrophils. HYPOTHESIS: Dexamethasone (DEX), a potent synthetic glucocorticoid, inhibits the functions of equine peripheral blood neutrophils through both genomic and non-genomic effects. ANIMALS: Six healthy adult mixed breed female horses. METHODS: To assess the genomic effects of DEX, peripheral blood neutrophils were isolated using a gradient technique and incubated 6 h with 100 ng/ml LPS and 10(-6) M DEX alone, or combined with the glucocorticoid receptor (GR) inhibitor RU486 (10(-5) M). Messenger RNA for IL-8, TNF-alpha and TLR-4 were measured using real-time RT-PCR. The non-genomic effects of DEX were studied in neutrophils incubated with 5 microM dichlorodihydrofluorescein (DCF) and 10(-6) M DEX 5, 10 and 15 min prior to being stimulated with 5 ng/ml phorbol myristate acetate. Neutrophils were similarly co-incubated with DEX (10(-6) M, 15 min) and RU486 (10(-5) M) to evaluate the contribution of the GR to these effects. The oxidation of DCF was studied using flow-cytometry. RESULTS: Neutrophils stimulation with LPS resulted in a significant increase in IL-8, TNF-alpha and TLR-4 mRNA expressions (p<0.0001); incubation with DEX significantly down-regulated this process (p<0.0001). DEX significantly reduced oxidation of DCF after 10 and 15 min of incubation (p<0.0001). Those effects were mediated through the GRs. CONCLUSION: DEX exerts anti-inflammatory effects on equine peripheral blood neutrophils through both genomic and non-genomic pathways.

Nebulisation of dexamethasone sodium phosphate for the treatment of severe asthmatic horses.

BACKGROUND: Inhaled corticosteroids are effective in the treatment of equine asthma. A recent study reported that nebulisation of injectable dexamethasone had low systemic bioavailability in healthy horses and could represent a cost-effective therapy for equine inflammatory lung diseases. OBJECTIVES: To determine the effects of dexamethasone nebulisation on lung function in severe asthmatic horses. It was hypothesised that dexamethasone administered by nebulisation would be more effective than the same dose administered orally. STUDY DESIGN: Randomised blinded experimental study in severe asthmatic horses. METHODS: Twelve severe asthmatic horses in clinical exacerbation were randomly assigned to treatment with 5 mg of dexamethasone sodium phosphate by nebulisation (n = 6) or by oral administration (n = 6), once daily for 7 days. Lung function was evaluated at baseline, after four treatment days (D4) and 24 h after the last dose (D8). The presence of residual bronchospasm was assessed on D8 with N-butylscopolammonium bromide administration (0.3 mg/kg i.v.). A respiratory clinical score was performed daily. Serum cortisol concentration was measured at baseline, D4 and D8. RESULTS: The pulmonary elastance was unchanged in both groups while pulmonary resistance was significantly improved in the oral group on D8 (mean reduction in 1 cm H2 O/L/s [CI: 0.34-1.65, P = 0.003]). All horses had residual bronchospasm at the end of the study. There was a group difference in the respiratory clinical score as it was significantly reduced in the oral group, from D5 to D8. Serum cortisol concentration decreased in all subjects. MAIN LIMITATIONS: Low number of horses and lack of placebo group. CONCLUSIONS: Considering the lack of improvement of lung function and the hypothalamic-pituitary-adrenal axis suppression, the results of this study do not support aerosolisation of an injectable form of dexamethasone for the treatment of severe equine asthma at the dose and with the nebuliser evaluated.

Effect of different doses of inhaled ciclesonide on lung function, clinical signs related to airflow limitation and serum cortisol levels in horses with experimentally induced mild to severe airway obstruction.

BACKGROUND: Inhaled corticosteroids are effective for the treatment of equine asthma but they induce cortisol suppression with potential side effects. OBJECTIVES: To study the efficacy of ciclesonide, an inhaled corticosteroid with an improved safety profile, on lung function, clinical signs related to airway obstruction, and serum cortisol levels in asthmatic horses exposed to a mouldy hay challenge. STUDY DESIGN: Cross-over placebo controlled, blinded, randomised experiment. METHODS: Sixteen horses were enrolled in three subsequent dose-titration studies (8 horses/study) to investigate the effects of inhaled ciclesonide administered for 2 weeks at doses ranging from 450 to 2700 µg twice daily or 3712.5 µg once daily. Systemic dexamethasone (0.066 mg/kg per os) was our positive control. A placebo group was also studied. Lung function and clinical scores were blindly performed before and after 7 and 14 days of treatment. Serum cortisol was measured before and after 3, 5, 7, 10, 14 days of treatment as well as 3 and 7 days post treatment. RESULTS: After 7 days, dexamethasone induced a significant reduction in pulmonary resistance (from 2.5 ± 0.6 at day 0 to 1.1 ± 0.7 cm H2 O/L/s), pulmonary elastance (5.0 ± 2.6 to 1.2 ± 1.0 cm H2 O/L), and of the weighted clinical score (14.8 ± 4.7 to 8.0 ± 4.4). Similarly, ciclesonide 1687.5 µg twice daily significantly improved pulmonary resistance (2.7 ± 1.1 to 1.6 ± 0.8 cm H2 O/L/s), pulmonary elastance (5.2 ± 3.1 to 2.2 ± 1.3 cm H2 O/L), and weighted clinical score (13 ± 2.9 to 10.8 ± 4.2). Serum cortisol suppression (<50 nmol/L) systematically occurred with dexamethasone from day 3 of treatment up to day 3 post treatment, but not with ciclesonide at any tested doses. Placebo did not exert any significant beneficial effect. MAIN LIMITATIONS: Experimentally induced asthma exacerbations in horses might respond differently to treatment than naturally occurring exacerbations. CONCLUSIONS: Inhaled ciclesonide is an effective treatment for horses with equine asthma. Serum cortisol was unaffected by treatment.

Efficacy of inhaled budesonide for the treatment of severe equine asthma.

BACKGROUND: Corticosteroids are the most potent drugs for the control of severe equine asthma, but adverse effects limit their chronic systemic administration. Inhaled medications allow for drug delivery directly into the airways, reducing the harmful effects of these drugs. OBJECTIVES: To evaluate the efficacy of inhaled budesonide specifically formulated for the equine use and administered by a novel inhalation device in horses with severe asthma. STUDY DESIGN: Experimental studies in horses with naturally occurring asthma with cross-over, randomised, blinded experimental designs. METHODS: In Study 1, budesonide (1800 µg twice daily) administered using a novel Respimat® based inhaler was compared to i.v. dexamethasone (0.04 mg/kg). In Study 2, 3 doses of budesonide (450, 900, and 1800 µg) were compared to oral dexamethasone (0.066 mg/kg). Lung function, bronchoalveolar fluid cytology (Study 1), CBC, serum chemistry, and serum cortisol and adrenocorticotropic hormone (ACTH) values were evaluated. RESULTS: In Study 1, there was a marked and significant improvement in the lung function of all horses treated with budesonide and dexamethasone. Neutrophil percentages in bronchoalveolar fluid decreased in all horses treated with dexamethasone and in four of six horses treated with budesonide. Serum cortisol and blood ACTH concentrations decreased with both treatments. In Study 2, there was a significant improvement in the lung function with all dosages of budesonide, and the effects of higher dosages were comparable to those of dexamethasone. Dexamethasone and budesonide at the two higher dosages induced a significant decrease of cortisol concentrations. MAIN LIMITATIONS: The Respimat® based inhaler is not currently commercially available. CONCLUSIONS: Administration of budesonide with the Respimat® based inhaler provided dose-dependent relief of airway obstruction in horses with severe asthma, but also a suppression of serum cortisol.

Report of the 3rd Havemeyer workshop on allergic diseases of the Horse, Hólar, Iceland, June 2007.

Allergic diseases occur in most mammals, although some species such as humans, dogs and horses seem to be more prone to develop allergies than others. In horses, insect bite hypersensitivity (IBH), an allergic dermatitis caused by bites of midges, and recurrent airway obstruction (RAO), a hyperreactivity to stable born dust and allergens, are the two most prevalent allergic diseases. Allergic diseases involve the interaction of three major factors: (i) genetic constitution, (ii) exposure to allergens, and (iii) a dysregulation of the immune response determined by (i) and (ii). However, other environmental factors such as infectious diseases, contact with endotoxin and degree of infestation with endoparasites have been shown to influence the prevalence of allergic diseases in humans. How these factors may impact upon allergic disease in the horse is unknown at this time. The 3rd workshop on Allergic Diseases of the Horse, with major sponsorship from the Havemeyer Foundation, was held in Hólar, Iceland, in June 2007 and focussed on immunological and genetic aspects of IBH and RAO. This particular venue was chosen because of the prevalence of IBH in exported Icelandic horses. The incidence of IBH is significantly different between Icelandic horses born in Europe or North America and those born in Iceland and exported as adults. Although the genetic factors and allergens are the same, exported adult horses show a greater incidence of IBH. This suggests that environmental or epigenetic factors may contribute to this response. This report summarizes the present state of knowledge and summarizes important issues discussed at the workshop.

Effects of a MAPK p38 inhibitor on lung function and airway inflammation in equine recurrent airway obstruction.

REASONS FOR PERFORMING STUDY: It has been suggested that many of the beneficial effects of corticosteroids are mediated through mitogen-activated protein kinase (MAPK) p38 inhibition. OBJECTIVE: To investigate the efficacy of the MAPK p38 inhibitor compound MRL-EQ1 to either prevent (Phase 1) or treat (Phase 2) recurrent airway obstruction (RAO) in horses. METHODS: MRL-EQ1 was administered i.v. at a dosage of 0.75-1.5 mg/kg bwt q. 12 h. In Phase 1, susceptible horses in clinical remission were divided into 2 groups (n = 5/group), based on historical values of respiratory mechanics. All horses were entered in the study in pairs (one control, one treated horse) and exposed to the same environmental challenge (stabling, mouldy hay and dusty conditions). The treatment group received MRL-EQ1 for 14 days while the control horses were untreated during the same period. In Phase 2, affected horses were ranked by severity of respiratory dysfunction and split randomly into either dexamethasone or MRL-EQ1 treatment groups (n = 5/group). Bronchoalveolar lavage fluid, respiratory mechanic measurements, MRL-EQ1 plasma concentration and tumour necrosis factor (TNF) whole blood activity were evaluated sequentially. RESULTS: In Phase 1, MRL-EQ1 did not prevent the occurrence of clinical signs and pulmonary inflammation. However, treatment was associated with a reduction in severity and a delay in the onset of signs and a reduction in pulmonary neutrophilia. In Phase 2, plasma concentrations achieved resulted in ex vivo suppression of lipopolysaccharide-induced TNF production in equine blood. MRL-EQ1 did not improve airway inflammation or lung function and was associated in a dose dependent manner with behavioural (depression, excitability) and blood changes (neutrophilia, increased serum muscle enzyme concentrations). CONCLUSIONS: Inhibition of p38 in the horse was partially effective in reducing clinical signs and airway inflammation when administered prior to, but not during clinical exacerbation in RAO. POTENTIAL RELEVANCE: Inhibitors of p38 MAPK with a better toxicity profile may be effective in the prevention or treatment of RAO.

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.

Age associated changes in peripheral airway smooth muscle mass of healthy horses.

Peripheral airway smooth muscle (ASM) mass is increased in severe equine asthma, but no information is available on age related changes in ASM. In this study, peripheral ASM dimensions were determined in healthy horses of different ages. The thickness of the peripheral ASM layer was constant in horses of different ages, but ASM occupied a greater proportion of the inner wall area in young horses compared to older horses. This finding suggests that equine airways experience a decrease in the relative abundance of ASM with age.

Theophylline does not potentiate the effects of a low dose of dexamethasone in horses with recurrent airway obstruction.

REASONS FOR PERFORMING STUDY: Theophylline has been shown to have corticosteroid-sparing effects for the treatment of human asthma. A similar effect, if present in horses, would allow diminishing the dose of corticosteroids administered to equine patients with inflammatory airway diseases. OBJECTIVES: To evaluate whether theophylline potentiates the effects of a low dose of dexamethasone when treating horses with recurrent airway obstruction (RAO). HYPOTHESIS: Theophylline has steroid-sparing effects in horses with RAO. METHODS: Ten mature mixed breed horses in clinical exacerbation of RAO were studied. Using an incomplete crossover design and 3 experimental periods of 7 days duration, horses were distributed randomly in 5 treatment groups; and administered dexamethasone s.i.d., at either 0.05 mg/kg bwt i.v. or per os, or 0.02 mg/kg bwt alone or combined with theophylline at 5 mg/kg bwt per os b.i.d. A fifth group was treated with theophylline alone at the above dosage. Lung function was evaluated prior to drug administration and then 3 and 7 days later. RESULTS: Oral administration of dexamethasone alone or combined with theophylline failed to improve lung function significantly in RAO affected horses. Theophylline alone also failed to improve lung function in all treated horses. Conversely, dexamethasone administration at 0.05 mg/kg bwt i.v. resulted in a significant improvement in lung function starting on Day 3. CONCLUSIONS AND POTENTIAL RELEVANCE: Oral theophylline for 7 days did not improve the effects of a low dose of dexamethasone for the treatment of horses with RAO.

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 (R5 : R10 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.

Environmental heat and airborne pollen concentration are associated with increased asthma severity in horses.

REASON FOR PERFORMING STUDY: Clinical exacerbations of severe equine asthma (formerly recurrent airway obstruction [RAO]) are more frequently reported during winter when horses are exposed to airborne dusts during stabling. However, we have also observed a worsening of clinical signs on days during a heatwave. OBJECTIVES: To investigate the association between environmental temperature and humidity and clinical signs of asthma in horses during clinical exacerbation of the disease. STUDY DESIGN: Retrospective longitudinal study. METHODS: Historical data on 14 severe asthmatic horses exposed to a dusty environment and evaluated using a previously validated clinical scoring system were analysed. Barn temperature and relative humidity values were obtained and air enthalpy (h) calculated. Correlation tests were used to study the relationship between mean daily clinical scores of horses and environmental variables. Lung function parameters recorded at 4 day intervals during hot (25°C) and warm (18°C) barn conditions were compared using a paired t test. RESULTS: Significant positive correlations were observed between the mean daily clinical score and temperature (r = 0.58, P = 0.01) and air enthalpy (r = 0.55, P = 0.02). Maximal daily temperature correlated with airborne pollen concentrations (r = 0.51, P = 0.0002). In the absence of changes in the management of horses, higher barn temperature and enthalpy were associated with increased transpulmonary pressure (P = 0.005), pulmonary resistance (P = 0.008) and elastance values (P = 0.005). CONCLUSIONS: Providing a cold environment may help attenuate the severity of airway obstruction in uncontrolled exacerbations of severe equine asthma. Furthermore, variations in environmental heat and associated pollen concentrations should also be taken into account when evaluating the response to therapy in clinical or research settings.

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.

Inflammatory Airway Disease of Horses--Revised Consensus Statement.

The purpose of this manuscript is to revise and update the previous consensus statement on inflammatory airway disease (IAD) in horses. Since 2007, a large number of scientific articles have been published on the topic and these new findings have led to a significant evolution of our understanding of IAD.

Physical training reverses defect in mitochondrial energy production in heart of chronically diabetic rats.

This study examined the impact of physical training on cardiac mitochondrial respiration of rats with chronic diabetes mellitus. Diabetes was induced by an intravenous injection of STZ (50 mg/kg) and only rats with a blood glucose level between 14 and 22 mM 1 wk later were kept in the protocol. Exercise training was conducted on a treadmill with a progressive 10-wk program. Animals were killed at the end of the training program, and mitochondria were isolated from ventricular tissue by differential centrifugation. Both state 3 respiration and oxidative phosphorylation rates were depressed significantly in the mitochondria of diabetic rats. These alterations were reversed completely to normal by physical training, without any significant changes in plasma glucose or insulin levels. The activity of ANT was not affected by diabetes or training. These results indicate that the depressed OPR present in isolated heart mitochondria from chronically diabetic rats is reversed to normal by physical training, apparently by mechanisms independent of blood glucose control. This correction in mitochondrial energy production may explain the improvement in cardiac function previously reported in trained diabetic rats.

Acute phase proteins in racehorses with inflammatory airway disease.

BACKGROUND: Systemic inflammation is observed in horses with heaves and could also be present in horses with a lesser degree of pulmonary inflammation. HYPOTHESIS/OBJECTIVES: It was hypothesized that racehorses with inflammatory airway disease (IAD) have increased concentration of circulating acute phase proteins. The objective of this study was to compare serum acute phase proteins of racehorses with and without lower airway inflammation. ANIMALS: Serum from 21 client-owned Standardbred racehorses with exercise intolerance and lower airway inflammation and serum from 10 client-owned Standardbred racehorses with exercise intolerance without lower airway inflammation. METHODS: In a case-control study, serum samples from previously characterized horses presented for exercise intolerance with or without lower airway inflammation based on bronchoalveolar lavage fluid cytology were analyzed for serum amyloid A protein (SAA), C-reactive protein (CRP), and haptoglobin using commercial ELISAs. RESULTS: There was no significant differences between groups for SAA (non-IAD versus IAD, median (range): 3.47 (0.06-34.94) versus 6.33 (0.06-80) µg/mL, P = .49), CRP (10.87 (2.05-29.03) versus 4.63 (0.02-31.81) µg/mL, P = .23) or haptoglobin (900.36 (607.99-2018.84) versus 749.54 (530.81-1076.95) µg/mL, P = .09). CONCLUSIONS AND CLINICAL IMPORTANCE: In this population of poorly performing racehorses in training, serum SAA, CRP, and haptoglobin were not helpful in distinguishing between horses with IAD from horses with exercise intolerance from other causes.