Degradation of Proteoglycans and Collagen in Equine Meniscal Tissues.

Investigate meniscal extracellular matrix degradation. Equine menisci (n = 34 from 17 horses) were studied. Site-matched sections were cut and scored from three regions (ROIs; n = 102) and stained for histology, proteoglycan (safranin O and fast green), aggrecan, and collagen cleavage (NITEGE, DIPEN, and C1,2C antibodies, respectively). Picrosirius red and second harmonic generation microscopy were performed to investigate collagen ultrastructure. A total of 42 ROIs met the inclusion criteria and were included in the final analysis. The median (range) ROI histological score was 3 (0-9), providing a large spectrum of pathology. The median (range) proteoglycan score was 1 (0-3), representing superficial and central meniscal loss. The median (range) of DIPEN, NITEGE, and C1,2C scores was 1 (0-3), revealing immunostaining of the femoral and tibial surfaces. The proteoglycan scores exhibited significant positive associations with both histologic evaluation (p = 0.03) and DIPEN scores (p = 0.02). Additionally, a robust positive association (p = 0.007) was observed between the two aggrecanolysis indicators, NITEGE and DIPEN scores. A negative association (p = 0.008) was identified between NITEGE and histological scores. The C1,2C scores were not associated with any other scores. Picrosirius red and second harmonic generation microscopy (SHGM) illustrated the loss of the collagen matrix and structure centrally. Proteoglycan and collagen degradation commonly occur superficially in menisci and less frequently centrally. The identification of central meniscal proteoglycan and collagen degradation provides novel insight into central meniscal degeneration. However, further research is needed to elucidate the etiology and sequence of degradative events.

Effects of soaked hay on lung function and airway inflammation in horses with severe asthma.

BACKGROUND: Reducing inhaled dust particles improves lung function in horses with severe asthma. Soaked hay is commonly used by owners, but its efficacy in improving lung function and inflammation has not been documented. OBJECTIVES: To measure the effects of soaked hay and alfalfa pellets in horses with severe asthma. ANIMALS: Ten adult horses with severe asthma from a research colony. METHODS: Prospective controlled trial. Horses in clinical exacerbation were housed indoors and allocated to be fed either soaked hay (n = 5) or alfalfa pellets (n = 5) for 6 weeks. Soaked hay was immersed for 45 minutes and dried out hay was discarded between meals. Pulmonary function and clinical scores were measured before and after 2, 4, and 6 weeks. Tracheal mucus scores and bronchoalveolar lavages were performed before and after 6 weeks. Lung function was analyzed with a linear mixed model using log-transformed data. RESULTS: Lung resistance decreased from (median (range)) 2.47 (1.54-3.95) to 1.59 (0.52-2.10) cmH2 O/L/s in the pellets group and from 1.89 (1.2-3.54) to 0.61 (0.42-2.08) cmH2 O/L/s in the soaked hay group over the 6-week period for an average difference of 1.06 cmH2 O/L/s for pellets (95% confidence interval [95% CI]: 0.09-2.04, P = .03, not significant after correction) and 1.31 cmH2 O/L/s for soaked hay (95% CI: -0.23 to 2.85, P < .001, significant). CONCLUSION AND CLINICAL IMPORTANCE: Soaked hay can control airway obstruction in horses with severe asthma. The strict protocol for soaking and discarding dried-out hay in this study could however be considered too great of an inconvenience by owners.

Characterization of fungal exposure and dectin-1 expression in healthy horses and horses with severe asthma.

OBJECTIVE: To quantify dectin-1 expression in bronchoalveolar lavage fluid (BALF), create polyclonal antibodies against equine dectin-1 and localize it in tissues, and quantify fungal exposure in pastured and stabled asthmatic and nonasthmatic horses. SAMPLES: BALF samples from 6 controls and 6 horses with severe asthma. Stored lung and nasal wash samples. PROCEDURES: Dectin-1 expression was quantified by quantitative PCR (qPCR). Purified peptide from equine dectin-1 was used to generate polyclonal antibodies and was confirmed with immunological testing. Fungal exposure was quantified in BALF samples by counting fungal-like intracellular particles in phagocytic cells, by qPCR quantification of the "universal" 18S rRNA fungal gene, and by quantifying 36 specific fungi in equine and dust samples using qPCR assays. RESULTS: Equine dectin-1 was localized in tissues and cells, and functional isoforms were upregulated significantly in BALF after stabling. Pastured horses from both groups had low levels of fungi in BALF, and there was a significant increase in some specific fungi, most notably for Eurotium amstelodami, Wallemia sebi, and Aspergillus niger after stabling. However, stabled asthmatic horses had fewer phagocytized particles, less 18S rRNA signal, and fewer specific fungi compared to nonasthmatic horses. CLINICAL RELEVANCE: Stabling increases exposure to fungi, but asthmatic horses had fewer fungi reaching their lower airways, presumably resulting from congestion and narrowing of the airways. Exposure to fungi could contribute to airway inflammation by increasing dectin-1 functional isoforms, and exposure to indoor molds should be avoided.

Assessment of thrombin generation in horses using a calibrated automated thrombogram.

BACKGROUND: The amount of thrombin generated reflects the endogenous thrombin potential (ETP), which depends on the balance of pro- and anticoagulant factors. The calibrated automated thrombogram (CAT) allows for the direct measurement of thrombin generation during the clotting process. OBJECTIVES: (1) To describe the results of the CAT assay in horses, (2) to establish intra-assay and intra- and interindividual variation of thrombin generation in healthy horses, and (3) to compare in vitro low-molecular-weight heparin (LMWH) sensitivity between healthy and sick horses. The hypothesis for the last objective is that inhibition of thrombin generation in sick horses requires higher heparin concentrations. METHODS: The plasma of 10 healthy mixed breed horses was used for the determination of normal thrombin generation parameters (lag time, time to peak, peak thrombin concentration, and ETP). Five of the healthy horses were compared with five horses with systemic inflammatory response syndrome (SIRS). In vitro heparin sensitivity was determined using LMWH. RESULTS: The intra-assay variation was small (<5%) for all parameters. Relatively large intra- and interindividual variation were observed in healthy horses. Four of the five sick horses with SIRS had a thrombogram compatible with a hypercoagulable state. The in vitro heparin sensitivity test suggested decreased sensitivity to LMWH in hypercoagulable states. CONCLUSIONS: The CAT assay could detect coagulopathy in horses. In vivo experiments are needed to confirm that it can be used to monitor responses to LMWH therapy.

Development of a computer-based quantification method for immunohistochemically-stained tissues and its application to study mast cells in equine wound healing (proof of concept).

BACKGROUND: There is a growing interest in the scientific community to use computer-based software programs for the quantification of cells during physiological and pathophysiological processes. Drawbacks of computer-based methods currently used to quantify immunohistochemical staining are the complexity of use, expense of software and overly-simplified descriptions of protocol thereby limiting reproducibility. The precise role of mast cells in equine cutaneous wound healing is unknown. Given the contribution of mast cells to the chronic inflammation observed in human keloid, a pathology similar to exuberant granulation tissue (EGT) in horses, mast cells might be present in high numbers in equine limb wounds predisposed to EGT. The main goal of this study was to develop a reliable and reproducible quantification method for immunostained tissues using a computer software that is widely available, at no cost, to the scientific community. A secondary goal was to conduct a proof of concept using the newly-established method to quantify mast cells during wound healing at different anatomical sites (body and limb) in horses to see if a different pattern is observed in limb wounds, which are predisposed to EGT. RESULTS: A good intraclass correlation coefficient (ICC, 0.67 p < 0.05) was found between the computer-based ImageJ method and manual counting. An excellent intra-operator ICC of 0.90 (p < 0.01) was found for the ImageJ quantification method while a good interoperator ICC of 0.69 (p < 0.01) was measured. No significant difference was observed between the variation of the ImageJ and that of the manual counting method. Mast cells were localized below the epidermis, around cutaneous appendages and blood vessels. Mast cell numbers did not differ significantly in relation to anatomical location or time of healing. CONCLUSIONS: The computer-based quantification method developed is reliable, reproducible, available, cost-free and could be used to study different physiological and pathological processes using immunohistochemistry.

Endothelial cell hypertrophy is associated with microvascular occlusion in horse wounds.

Wound repair in horse limbs is often complicated by excessive fibroplasia and scarring. Occlusion of the microvessels populating the granulation tissue appears to be involved in the excessive accumulation of extracellular matrix during the repair of limb wounds. This study aimed to determine whether endothelial cell hypertrophy or hyperplasia, or both, contribute to microvascular occlusion and whether the pericyte is involved in this anomaly. We created 5 wounds, each 2.5 x 2.5 cm, on both forelimbs and on the body of 6 horses. One limb was bandaged to stimulate excessive wound fibroplasia. Weekly biopsy specimens were evaluated by transmission electron microscopy to measure microvessel luminal diameters and the surface area of endothelial cells and to count endothelial cells and pericytes. Microvessels were occluded significantly more often in limb wounds than in body wounds. The surface area of endothelial cells lining occluded microvessels (mean +/- standard error, 28.4013 +/- 1.5154 microm2) was significantly greater (P = 0.05) than that of cells lining patent microvessels (26.2220 +/- 1.5268 microm2). Conversely, neither the number of endothelial cells nor the number of pericytes differed between patent and occluded microvessels or between limb and body wounds. Furthermore, the wound location and the status of the microvessels (patent or occluded) did not alter the ratio of endothelial cells to pericytes. These data suggest that endothelial cell hypertrophy might play a role in the microvascular occlusion present in granulation tissue of limb wounds in horses, but the contribution of the pericyte remains obscure.