Heaves-affected Horses Research Articles

Evaluation of contractile phenotype in airway smooth muscle cells isolated from endobronchial biopsy and tissue specimens from horses.

OBJECTIVE To develop a method to maintain the initial phenotype of airway smooth muscle (ASM) cells isolated from equine endobronchial biopsy specimens in long-term cell culture. SAMPLE Endobronchial tissue specimens (8 to 10/horse) collected from the lungs of previously healthy horses at necropsy (n = 12) and endobronchial biopsy specimens collected from standing, sedated, heaves-affected horses in clinical remission of the disease (5) and control horses (4). PROCEDURES A sampling protocol was developed to recover and maintain a contractile phenotype in ASM cells from endobronchial specimens from freshly harvested equine lungs and from healthy and heaves-affected horses. Immunologic techniques were used to evaluate the contractile phenotype of ASM cells in culture. RESULTS Characteristic ASM cells were successfully cultured from endobronchial tissue or biopsy specimens from both healthy and heaves-affected horses, and their contractile phenotype was maintained for up to 7 passages. Moreover, the capacity of cells at the seventh passage to contract in a collagen gel in response to methacholine was maintained. CONCLUSIONS AND CLINICAL RELEVANCE ASM cells isolated from equine endobronchial tissue and biopsy specimens were able to maintain a contractile phenotype in long-term cell cultures, suggesting they could be used for tissue engineering and in vitro studies of equine ASM cells.

Peripheral Airway Smooth Muscle, but Not the Trachealis, Is Hypercontractile in an Equine Model of Asthma.

Heaves is a naturally occurring equine disease that shares many similarities with human asthma, including reversible antigen-induced bronchoconstriction, airway inflammation, and remodeling. The purpose of this study was to determine whether the trachealis muscle is mechanically representative of the peripheral airway smooth muscle (ASM) in an equine model of asthma. Tracheal and peripheral ASM of heaves-affected horses under exacerbation, or under clinical remission of the disease, and control horses were dissected and freed of epithelium to measure unloaded shortening velocity (Vmax), stress (force/cross-sectional area), methacholine effective concentration at which 50% of the maximum response is obtained, and stiffness. Myofibrillar Mg(2+)-ATPase activity, actomyosin in vitro motility, and contractile protein expression were also measured. Horses with heaves had significantly greater Vmax and Mg(2+)-ATPase activity in peripheral airway but not in tracheal smooth muscle. In addition, a significant correlation was found between Vmax and the time elapsed since the end of the corticosteroid treatment for the peripheral airways in horses with heaves. Maximal stress and stiffness were greater in the peripheral airways of the horses under remission compared with controls and the horses under exacerbation, potentially due to remodeling. Actomyosin in vitro motility was not different between controls and horses with heaves. These data demonstrate that peripheral ASM is mechanically and biochemically altered in heaves, whereas the trachealis behaves as in control horses. It is therefore conceivable that the trachealis muscle may not be representative of the peripheral ASM in human asthma either, but this will require further investigation.

Blood hypercoagulability and systemic inflammation in horses with heaves

As inflammation and coagulation are intertwined processes, the efficiency of blood coagulation of heaves-affected horses and controls were compared in an observational case-control study, using thrombelastography. In experiment 1 (Exacerbation, six heaves, five controls), horses were housed indoors and fed hay. Thrombelastography, functional fibrinogen, platelet count, hematology, and antithrombin were measured. In experiment 2 (Remission, eight heaves, 11 controls), horses were housed in a low-dust environment for at least a month when thrombelastography was performed. Heaves-affected horses in exacerbation had greater maximum amplitude and higher functional fibrinogen than controls. Heaves-affected horses in clinical remission had greater maximum amplitude than controls. The hypercoagulable state and systemic inflammation of horses with heaves may be a consequence of pulmonary inflammation and may contribute to the perpetuation of airway dysfunction.

Bronchial but not Tracheal Smooth Muscle is Hypercontractile in an Equine Model of Severe Asthma

Asthma is an inflammatory disease of the airways characterized by airway hyperresponsiveness, an exaggerated bronchoconstrictive response of airway smooth muscle (ASM) to stimuli. However, whether asthmatic ASM is mechanically different from non-asthmatic ASM is still unclear. Most studies so far have been performed on the trachealis muscle but it might not be representative of smooth muscle in the more peripheral airways. Thus, we addressed this question in an equine model of asthma (horse with heaves) and compared the trachealis (TSM) and peripheral bronchial (BSM) airway smooth muscle mechanics.Muscle strips from de-epithelialized TSM and BSM (less than 10 mm in diameter) were mounted horizontally in an experimental chamber between a length controller and a force transducer (Aurora Scientific Inc.). Maximal velocity of shortening (Vmax), methacholine dose response and stress were measured.We found no difference in Vmax between TSM from control horses and horses with heaves (0.124±0.01vs. 0.119±0.01; control vs. heaves; P=0.8), whereas the BSM exhibited a significantly higher Vmax (0.114±0.01 vs. 0.239±0.02; P=0.03). Western blot analysis showed a significant increase of the (+)insert fast smooth muscle myosin heavy chain (SMMHC) isoform in BSM of heaves-affected horses (0.78±0.04 vs. 1.0±0.05, P=0.0092), while no such difference was found in TSM (0.91±0.05 vs.0.97±0.08;P=0.54). The expression of calponin was significantly increased in TSM of heaves-affected horses (0.92±0.15 vs. 2.5±0.57, P=0.037; but this was not observed in BSM (0.37±0.14 vs. 0.49±0.18; P=0.62). There were no differences detected in the expression of MLCK, total SMMHC and transgelin. Peripheral airways may have a greater exposure to inflammatory cells than the trachealis which may explain their differences in mechanics. Further studies await to determine the mechanism underlying in observed effect.Supported by: CIHR(MOP102718), NIH(RO1HL103405), RFBR(12-04-0076)

Serum cortisol concentration in horses with heaves treated with fluticasone proprionate over a 1 year period

The purpose of this study is to measure the effect of long-term administration of inhaled fluticasone proprionate on cortisol concentrations in heaves-affected horses. Eleven horses with heaves were treated with fluticasone at least once daily at dosages required to improve lung function or with antigen avoidance alone for 1 year. Morning serum cortisol was measured before and after 10, 30, 110, 190, 230, 280, and 320 days of treatment. Cortisol was also measured in the afternoon of day 330. Cortisol was significantly lower in the Fluticasone group on days 30, 110, and 190 when compared with the Antigen avoidance group. Cortisol measured on day 330 was also significantly lower in the Fluticasone group. Results indicate that inhaled fluticasone, when administered at therapeutic dosages, can significantly suppress serum cortisol concentrations for 8–24 h. The clinical significance of this finding remains to be ascertained, as no clinical signs were associated with this cortisol suppression.

Circulating immune complexes and markers of systemic inflammation in RAO-affected horses.

The aim of this study was to investigate the levels of circulating immune complexes (CICs) and concentration of haptoglobin, fibrinogen and C-reactive protein in the serum of horses with recurrent airway obstruction and healthy controls. The study was conducted on a group of 14 adult Polish Konik horses, kept in uniform environmental and living conditions. Horses were divided into two groups: 7 horses were not affected by any respiratory problem (control group) and 7 horses had a history of recurrent airway obstruction (RAO) (study group). A clinical and laboratory evaluation, endoscopic examination and bronchoalveolar lavage (BAL) were performed in all horses. Levels of circulating immune complexes were significantly (p = 0.0057) increased in heaves-affected horses compared to healthy controls (median [25th-75th percentiles]) (3.96 [3.96-4.43] vs. 7.46 [5.13-11.9]). No significant difference was observed in the levels of the examined acute phase proteins between the groups. Moreover, all results were within the reference range established for horses. The results of this study indicate that heaves in horses is associated with the formation and high level of CICs. Haptoglobin, fibrinogen and C-reactive protein failed as markers of early stage systemic inflammation in the course of RAO.

Inhaled corticosteroids modulate the (+)insert smooth muscle myosin heavy chain in the equine asthmatic airways.

Overexpression of the (+)insert smooth muscle myosin heavy chain (SMMHC) isoform could contribute to airway bronchospasm by increasing the velocity of contraction. Whether the (+)insert isoform is present in the small airways and its expression is reversible in asthma are unknown. To determine the anatomical location and the expression kinetics of the (+)insert SMMHC isoform in airways of horses with heaves and to evaluate its modulation in response to disease status. We evaluated the (+)insert SMMHC isoform in the airways of horses with heaves during disease exacerbation and remission, and in controls. The expression kinetics of the SMMHC (+)insert was then assessed at multiple time points in two studies: first, in horses with heaves treated for a 1-year period with antigen avoidance alone, inhaled corticosteroids alone or both; second, in horses with heaves before and after a 30-day natural antigen exposure. Gene expression analysis was assessed by quantitative PCR and protein expression was confirmed by targeted mass spectrometry. The (+)insert SMMHC isoform was significantly increased in central and peripheral airways, but not in the trachea of heaves-affected horses in clinical exacerbation when compared horses with heaves in remission and controls. Both corticosteroid administration and antigen avoidance led to a significant reduction of the (+)insert expression in the airways. The (+)insert SMMHC isoform was not significantly increased in airways after 1 month of antigenic re-exposure. The (+)insert SMMHC expression is increased throughout the bronchial tree in horses with heaves and reversible by corticosteroids administration and antigen avoidance.

Airway wall eosinophilia is not a feature of equine heaves

The objective of this study was to determine whether eosinophils infiltrate the airway wall of horses with heaves. Eosinophils were evaluated using paraffin embedded lung tissues from six heaves-affected horses in crisis and six aged-matched controls. Slides were stained using Luna's method and eosinophils enumerated using histomorphometric techniques. Total eosinophil counts (expressed per mm2 of basement membrane) were significantly higher in the airways of controls horses than in horses with heaves. Intraluminal, intraepithelial, and airway smooth muscle eosinophils counts were also increased in control horses. The results suggest that eosinophils do not contribute to the persistent airway obstruction 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.

Airway collagen and elastic fiber content correlates with lung function in equine heaves.

The consequences on lung function and inflammation of alterations in the extracellular matrix affecting the peripheral airway wall in asthma are largely unknown. We hypothesized that remodeling of collagen and elastic fibers in the peripheral airway wall leads to airway obstruction and contributes to neutrophilic airway inflammation. Animals used were six heaves-affected horses and five controls. Large peripheral lung biopsies were obtained from horses with heaves in clinical remission (Baseline) and during disease exacerbation and from age-matched controls. The area of collagen and elastic fiber content in the lamina propria was measured by histological staining techniques and corrected for airway size. Collagen type 1 and type 3 content was further assessed from additional horses after postmortem lung samples by immunohistochemistry. The collagen breakdown products proline-glycine-proline (PGP) and N-acetylated-PGP (N-α-PGP) were also measured in bronchoalveolar lavage fluids (BALF) by mass spectrometry. Compared with controls, heaves-affected horses had an increase in collagen (P = 0.05) and elastic fiber contents (P = 0.04) at baseline. Collagen types 1 and 3 content was also significantly increased in diseased horses (P = 0.015) when both collagen types were combined. No further change in collagen content was observed after a 30-day antigenic challenge. Airway collagen at baseline was positively correlated with pulmonary resistance in asthmatic horses (r(2) = 0.78, P = 0.03) and elastic fiber content was positively associated with pulmonary elastance in controls (r(2) = 0.95, P = 0.02). No difference between groups was appreciated in PGP and N-α-PGP peptides in BALF. Increased airway wall collagen and elastic fiber content may contribute to residual obstruction in the asthmatic airways.

Proteomic analysis of bronchoalveolar lavage fluid in an equine model of asthma during a natural antigen exposure trial

Background Heaves is a complex, asthma-like respiratory disease that affects many older horses. While environmental and genetic components to the disease have been proposed, the specific pathophysiology of heaves is still poorly understood. Using proteomic techniques, we compared the protein profile of bronchoalveolar lavage fluid (BALF) in the lungs of healthy horses and horses affected with heaves. Methods Clinical signs of the disease were induced in heaves-affected horses using an experimental hay exposure model. Samples of BALF were collected from all horses before and after the hay exposure trial. Mass spectrometry (LC-MS) was used to evaluate the differences in the global BALF peptide profile between the control and heaves-affected horses. Tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed proteins in the two groups of horses. The identification of two proteins was validated with Western blot assays. Results One hundred peptides were differentially expressed between healthy controls and heaves-affected horses; 76 peptides were over-expressed in controls and 24 were over-expressed in heaves-affected horses. The identifications of transferrin and secretoglobin were confirmed with Western blot. Conclusions This study demonstrates that proteomics can be used to compare the protein profiles of BALF from healthy and diseased horses. These techniques may prove helpful in determining the pathophysiology of complex diseases.

PI3K andMAPKs Regulate Neutrophil Migration Toward the Airways in Heaves

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. MAPK and PI3K pathways are involved in neutrophil migration toward the airway lumen in heaves. Twelve heaves-affected horses and 4 healthy horses. 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. 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. 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.

Markers of Systemic Inflammation in Horses with Heaves

Systemic inflammation in horses with heaves is poorly characterized. To assess acute phase proteins (APP) and inflammatory cytokine profiles in serum of healthy horses and horses with heaves. Six healthy horses and 6 heaves-affected horses belonging to the University of Montreal. 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. 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. In heaves, haptoglobin is a marker of both acute and chronic systemic inflammation, whereas high concentrations of SAA indicate acute inflammation.

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.

Systemic inflammation and priming of peripheral blood leukocytes persist during clinical remission in horses with heaves

ObjectiveTo compare innate immune responses of peripheral blood leukocytes from healthy and asymptomatic heaves-affected horses. AnimalsHeaves-affected horses (n=5-6) and healthy controls (n=4–5) kept under low dust environments (pasture or shavings and pellets). MethodsBlood neutrophil and neutrophil-depleted cell populations were isolated using MACS system. Cells were incubated with or without bacterial products (lipopolysaccharide (LPS), 100ng/mL and fMLP, 5ng/mL, 5h). Cytokine (IL-1β, IL-8, TNF, IL-4, INFγ and IL-10) and receptor (TLR4) mRNA expression was assessed by qPCR. TNF concentration in culture supernatants and serum samples was assessed using equine specific ELISA. Apoptotic rate of resting and stimulated neutrophils was assessed by flow cytometry using AnnexinV and 7-AAD (18h) and correlated with early pro-inflammatory cytokine expression in the same cells (5h). ResultsStimulation with bacterial-derived products resulted in overexpression of pro-inflammatory cytokines in both neutrophils (IL-1β and TNF) and neutrophil-depleted leukocytes (IL-1β and IL-8) from heaves-affected horses. Neutrophil survival (18h) was associated with their early TNF expression, but not IL-8. Neutrophil-depleted leukocytes from these horses also had significantly increased basal TNF mRNA levels. Serum TNF concentration was also significantly higher in heaves-affected horses compared to healthy horses kept in similar environment. ConclusionsAltered innate immune response to bacterial products is observable ex vivo in peripheral blood leukocytes from asymptomatic heaves-susceptible horses and is associated with high serum TNF concentration. It remains to be determined if this phenomenon is caused by intrinsic differences in innate immune responses or to cellular priming caused by systemic inflammation.

Profiling of Differentially Expressed Genes Using Suppression Subtractive Hybridization in an Equine Model of Chronic Asthma

BackgroundGene 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.ObjectiveTo identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition.MethodsEleven 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.ResultsHorses 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.ConclusionsPathways 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.

Effect of Long‐Term Fluticasone Treatment on Immune Function in Horses with Heaves

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. To evaluate the effects of prolonged administration of inhaled fluticasone on the immune system of heaves-affected horses. Heaves-affected horses were treated with inhaled fluticasone (n = 5) for 11 months or received environmental modifications only (n = 5). 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. No clinical adverse effects were observed and no differences in immune function were detected between treated and untreated horses. 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.

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.

Thoracoscopic Lung Biopsies in Heaves-Affected Horses Using a Bipolar Tissue Sealing System

To validate the use of the LigaSure™ Vessel Sealing System (LVSS) to perform thoracoscopic lung tissue biopsies in heaves-affected horses. Prospective clinical study. Heaves-affected horses (n=12). 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. 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. 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. LVSS can be used with relative safety to perform thoracoscopic lung biopsy, but close postoperative monitoring is necessary to avoid tension pneumothorax.

Effect of composition and different fractions of hay dust suspension on inflammation in lungs of heaves-affected horses: MMP-9 and MMP-2 as indicators of tissue destruction

Airway matrix metalloproteinases (MMPs) increase following inhalation of organic dust. The relative contribution of dust components to this elevation is unknown. To identify components of organic dust responsible for elevated MMP levels in equine airways. Bronchoalveolar lavage (BALF) from 7 heaves-susceptible horses, collected 6 h following inhalation challenges with saline, 2 different hay dust suspensions (HDS-1 and -2) and soluble and particulate fractions of HDS-1, were analysed for MMP-2 and -9 using SDS-page gelatin zymography. HDS-1 challenge increased BALF proMMP-9 and total MMP-9. HDS-1 fractions, or the particulate fraction with added lipopolysaccharide, increased BALF proMMP-9 and total MMP-9 in combination, but not when inhaled separately. HDS-2 inhalation elevated BALF complex forms, proMMP-9, active MMP-9, total MMP-9 and total MMP-2. The results suggest synergistic action of soluble and particulate organic dust components. The fact that HDS-1 and HDS-2 had different glucan concentrations supports a role for moulds in the activation of MMP-9. Activation and release of MMPs in response to inhaled moulds are involved in the aetiopathogenesis of heaves. Endotoxin contributes to the synergistic action of the dust components, but the overall MMP response to organic dust inhalation in heaves-susceptible horses largely reflects the mould content of the dust. In the future, inhibition of MMP production and release may offer therapeutic means for treatment and prevention of heaves and recommendations for acceptable dust levels can be given.