Characterisation of IgE-mediated histamine release from equine basophils in vitro

In vitro IgE-mediated histamine release by equine blood basophils was characterised as the basis for a screening test for immediate hypersensitivity responses in horses. The responses are initiated by inducing agents that are capable of crosslinking or bridging the membrane-bound IgE molecules. The release process is complete within 40 mins. In vitro histamine release is dose-dependent, with a submaximal response at less or greater than the optimal dose of inducing agent. Exogenous calcium is required but not magnesium; the optimal release calcium concentration is 1.0 to 1.5 mM. If an IgE-mediated inducing agent is added in the absence of exogenous calcium, the basophils become desensitised. The pH and temperature optima for release are physiological (pH 7.4, 37 degrees C). Histamine release is potentiated by deuterium oxide.     

In vitro dose-dependent effects of enrofloxacin on equine articular cartilage.

OBJECTIVE: To determine whether enrofloxacin has detrimental, dose-dependent effects on equine articular cartilage in vitro. ANIMALS: Cartilage explants were developed from 6 healthy horses between 0 and 96 months old. PROCEDURE: Patellar cartilage explants were incubated in 5 concentrations of enrofloxacin (2 microg/ml, 10 microg/ml, 1,000 microg/ml, 10,000 microg/ml, and 50,000 microg/ml) for 72 hours. Proteoglycan synthesis (Na35SO4 incorporation for 24 hours), proteoglycan degradation (Na35SO4 release for 72 hours), endogenous proteoglycan content (dimethylmethlene blue assay), and total protein content were determined. Cartilage explants were evaluated by use of histomorphologic and histomorphometric techniques (toluidine blue stain) for cytologic and matrix characteristics. Quantitative data were analyzed with a one-way ANOVA to compare results among various enrofloxacin concentration groups and the control group. A general linear model was used to determine whether age had an effect. RESULT: Proteoglycan synthesis was excellent in control specimens and in specimens incubated in low concentrations of enrofloxacin (2 microg/ml and 10 microg/ml). High concentrations of enrofloxacin (> 1,000 microg/ml) effectively eliminated proteoglycan synthesis regardless of horse age. Proteoglycan degradation at low concentrations (2 microg/ml and 10 microg/ml) was not different than control. High concentrations of enrofloxacin (> 1,000 microg/ml) caused significant degradation. Different concentrations of enrofloxacin did not affect endogenous proteoglycan. High concentrations of enrofloxacin were associated with a significant increase in number of pyknotic nuclei. CONCLUSION: Concentrations of enrofloxacin that might be achieved following systemic administration did not suppress chondrocyte metabolism in vitro. High concentrations of enrofloxacin (> 1,000 microg/ml) were toxic to chondrocytes.     

Characterisation of lymphocyte subsets in the equine oviduct

REASONS FOR PERFORMING STUDY: The equine oviduct is the site of fertilisation and location of embryonic development during the first 5 or 6 days. It therefore has an important influence on mare fertility. Although histopathological changes have been described previously, there is limited information regarding lymphocyte subtypes present in the mucosa of the normal equine oviduct. OBJECTIVES: To characterise the distribution of CD3+, CD4+, CD8+ and B lymphocytes in the equine oviduct from inseminated mares during oestrus and dioestrus, and from noninseminated mares during the immediate post ovulatory period. METHODS: Oviductal tissues were collected from noninseminated mares at oestrus (> 30 mm follicle, n = 4), at Day 1 post ovulation (n = 3) and at dioestrus (Day 7 post ovulation; n = 4). Oviducts were also collected from inseminated mares at Days 1, 2, and 3 post ovulation (n = 4 for each period). Cross-sections of tissues from the ampullar-isthmic junction from each oviduct were snap frozen and cryostat sections stained by the immunoperoxidase technique with monoclonal antibodies directed against equine lymphocyte surface markers for B cells as well as CD3+, CD4+ and CD8+ cells. RESULTS: In all oviductal sections examined, B cells were rare whereas T cells were relatively abundant. The predominant cell type found was the CD8+ phenotype, with a lesser number of CD4+ cells. Among mares, individual variation was large; therefore, although breeding status and stage of oestrous cycle appeared to alter lymphocyte populations, these differences were not significant. CONCLUSIONS AND POTENTIAL RELEVANCE: A population of CD3+, CD4+ and CD8+ cells exists within the mucosal region of the equine oviduct. The density of these cells is similar to that described in the human oviduct. Their function is not currently known, but they may be involved with modulation of the maternal response to the presence of spermatozoa or the early conceptus within the equine oviduct. As our capacity to differentiate these cell types improves, along with the ability to identify the specific cytokines they produce, their functional significance will become more apparent.     

Variations in cartilage catabolism in different equine joints in response to interleukin-1 in vitro

An explant system was used to investigate the hypothesis that cartilage from different equine joints might respond differently to challenge with interleukin-1alpha (IL-1alpha). Pairs of normal cartilage samples were taken from the metacarpophalangeal, proximal interphalangeal and distal interphalangeal joints of six horses. One of each pair was stimulated with 10 ng/ml human recombinant IL-1alpha for three days, and the supernatants and remaining cartilage explants were analysed for their total content of glycosaminoglycans. A significantly higher percentage of glycosaminoglycans was released from the cartilage of the proximal and distal interphalangeal joints than from the metacarpophalangeal joint.     

The effects of radial shock waves on the metabolism of equine cartilage explants in vitro

AIM: To investigate, in vitro, the effects of radial shock waves on the release of nitric oxide (NO) and synthesis of prostaglandin E2 (PGE2) and glycosaminoglycan (GAG), and liberation of GAG, from equine articular cartilage explants. METHODS: Equine cartilage from normal metacarpophalangeal and metatarsophalangeal joints was exposed to radial shock waves at various impulse doses and then maintained as explants in culture for 48 h. Shock waves were delivered at 1,876 Torr pressure and a frequency of 10 Hz. Treatment groups consisted of a negative control group, or application of 500, 2,000, or 4,000 impulses by use of either a convex handpiece (Group A) or concave handpiece (Group B). Synthesis of GAG was measured using incorporation of 35S-labelled sodium sulphate. Additionally, the synthesis of NO and PGE2, and content of GAG of the explants and media were determined. RESULTS: No significant effects (p>0.05) of radial shock-wave treatment were evident on the synthesis of NO or PGE2, or release of GAG by cartilage explants. However, radial shock waves decreased synthesis of GAG measured 48 h after exposure for all treatment groups other than the 500-impulse Group-A explants (p<0.05). CONCLUSIONS: Radial shock waves impact the metabolism of GAG in chondrocytes in equine articular cartilage. Further studies will be required to fully investigate the impact of this effect on the health of joints, and to elucidate the clinical impact.     

Characterisation and quantification of equine interferon gamma

Interferon-gamma (IFN-gamma) is a key cytokine in cell-mediated immunity. To measure IFN-gamma production of equine lymphocytes (eqIFN-gamma), we developed a quantitative ELISA. Monoclonal antibodies (mAb) were produced against bacterially derived eqIFN-gamma. The mAbs recognised recombinant and lymphocyte-derived eqIFN-gamma in ELISA, Western blotting, as well as flow cytometric and microscopic analysis. In contrast to bacterially derived material, mammalian and insect cell-derived eqIFN-gamma was biologically active but could be neutralised by one of the monoclonal antibodies. Unexpectedly, glycosylation seemed to be required for antiviral activity of eqIFN-gamma.     

In vitro effect of ventriculocordectomy before laryngoplasty on abduction of the equine arytenoid cartilage

Objective: To determine whether ventriculocordectomy (VCE) performed before prosthetic laryngoplasty (PL) results in increased rima glottidis size compared with PL alone. Study Design: Experimental study. Animals: Equine cadaver larynges (n=13). Methods: Right arytenoid cartilages were maximally abducted using a standard PL technique. Standard PLs were then performed on the left side and the force required to maximally abduct the left arytenoid cartilage recorded (F_max). Photographs were taken of the rima glottidis at zero force and at five equal levels of force up to F_max. The force applied was released, left VCE performed, and photographs repeated. Arytenoid left:right angle quotients (LRQ) and glottic cross‐sectional area ratios (CSAR) were calculated at each force level in each condition (PL and VCE‐PL). Results: Mean LRQ and CSAR for both PL and VCE‐PL increased with increasing force, initially rapidly before plateauing at ～50% of F_max. LRQ and CSAR were significantly greater for VCE‐PL than for PL (P<.001). When VCE was performed before PL, 12% less force was required to achieve an LRQ of 0.8, and 45% less for a CSAR of 0.8. Conclusions: In vitro, VCE performed before PL enables the arytenoid cartilage to be abducted to a greater degree for a given PL suture force.     

Age-related morphometry of equine calcified cartilage

Although there are many studies in the equine literature focused on articular diseases and the aetiology of osteoarthritis, few have concentrated on normal articular structures and how they change with age. The objective of this investigation was to study the thickness and morphology of the calcified cartilage layer of the distal metacarpus over a range of ages. A parasagittal slab of bone was sectioned from the region of sesamoid contact on the medial condyle of the metacarpi from 34 horses. The slab of bone was preserved, dehydrated and embedded, undecalcified, in methylmethacrylate and then stained with toluidine blue. Six repeatable fields of interest from the distal aspect of each metacarpus were digitised and examined to determine the morphology of the calcified cartilage layer. The thickness of the calcified cartilage, range 88-426 microm, was estimated using a method of integration. The results indicate an age-related influence on the thickness of the calcified cartilage layer, generally increased in older horses. While this finding is significant, perhaps more importantly a positional relationship was also identified, indicating that pressures endured by different regions within a joint may dictate morphological development of the tissues. This study has begun to lay the groundwork to determine whether the calcified layer of the hyaline cartilage could be involved in the development of osteoarthritis.     

Biomechanical characterisation of equine laryngeal cartilage

Reasons for performing the study: Upper airway obstruction is a common problem in the performance horse as the soft tissues of the larynx collapse into the airway, yet there is a paucity of information on biomechanical properties for the structural cartilage components. Objective: To measure the geometry and compressive mechanical properties of the hyaline cartilage to improve understanding of laryngeal function and morphology. Methods: A total of 11 larynges were harvested from Thoroughbred and Standardbred racehorses. During gross dissection, linear dimensions of the cricoid were obtained. From both the cricoid and arytenoid, specimens were cored to obtain 6 mm disc samples from 3 sites within the dorsal cricoid (caudal, middle and rostral) and 2 central sites in the arytenoids (inner, outer). The specimens were mechanically tested using radial confined compression to calculate the aggregate modulus and permeability of the tissue. The biomechanical data were analysed using a nested mixed effects model. Results: Geometrically, the cricoid has relatively straight walls compared to the morphology of human, ovine and canine larynges. There were significant observations of higher modulus with increasing age (0.13 MPa per year; P = 0.007) and stiffer cricoid cartilage (2.29 MPa) than the arytenoid cartilage (0.42 MPa; P<0.001), but no difference was observed between the left and right sides. Linear contrasts showed that the rostral aspect (2.51 MPa) of the cricoid was 20% stiffer than the caudal aspect (2.09 MPa; P = 0.025), with no difference between the arytenoid sites. Conclusions: The equine larynx is a well supported structure due to both the geometry and material properties of the cricoid cartilage. The hyaline structure is an order of magnitude higher in compressive modulus compared to the arytenoids and other hyaline‐composed tissues. Potential relevance: These characterisations are important to understand the biomechanics of laryngeal function and the mechanisms involved with surgical interventions.     

In vitro stimulation of equine articular cartilage proteoglycan synthesis by hyaluronan and carprofen.

The effects of hyaluronan and carprofen (both racemic mixture and separate R and S enantiomers) on proteoglycan (PG) synthesis by equine cultured chondrocytes and cartilage explants were examined. Hyaluronan stimulated PG synthesis in both cell and explant cultures. The concentration-response curve of the latter was bell-shaped. Racemic carprofen and R and S enantiomers also stimulated PG synthesis, although concentration-response relationships varied for each preparation and high concentrations inhibited synthesis. It was concluded that (a) hyaluronan exerts a stimulatory effect on PG synthesis at low concentrations and (b) stimulatory effects of carprofen on PG synthesis are, to some degree, enantioselective with the carprofen S-enantiomer exerting the greatest effect. Hyaluronan and carprofen are used clinically despite incompletely understood mechanisms of action. These results suggest (a) hyaluronan and carprofen might exert an anti-arthritic action through stimulation of PG synthesis and (b) there is possible justification for therapeutic administration of enantiomeric rather than racemic carprofen.     

In vitro evaluation of the effect of dimethyl sulfoxide on equine articular cartilage matrix metabolism

OBJECTIVE: To evaluate the effects of dimethyl sulfoxide (DMSO) on equine articular cartilage matrix metabolism. STUDY DESIGN: Using a cartilage explant culture system, proteoglycan (PG) synthesis, PG release, lactate metabolism, chondrocyte viability, and metabolism recovery were determined after cartilage exposure to DMSO. SAMPLE POPULATION: Cartilage harvested from metacarpophalangeal and metatarsophalangeal joints of 12 horses (age range, 1 to 10 years). METHODS: Explants were exposed to concentrations of DMSO (1% to 20%) for variable times (3 to 72 hours). PG synthesis and release were determined by a radiolabel incorporation assay and dimethylmethylene blue (DMMB) dye assay, respectively. Lactate released into culture media was measured, and chondrocyte viability was assessed using the Formizan Conversion Assay and a paravital staining protocol. Metabolism recovery was assessed in explants that were allowed to recover in maintenance media after exposure to DMSO. RESULTS: PG synthesis and lactate metabolism were inhibited in a dose- and time-dependent manner after exposure to DMSO concentrations > or = 5%; there was no significant alteration in PG release. No change in chondrocyte viability was detected after incubation with DMSO. PG synthesis and lactate metabolism returned to baseline rates when allowed a recovery period after exposure to DMSO. CONCLUSIONS: DMSO concentrations > or = 5% suppress equine articular cartilage matrix metabolism. Suppression of PG synthesis and lactate metabolism is reversible and does not appear to be the result of chondrocyte death. CLINICAL RELEVANCE: Equine clinicians adding DMSO to intraarticular lavage solutions should be aware that DMSO may have deleterious effects on equine articular cartilage matrix metabolism.     

Assessment of cartilage degradation effects of matrix metalloproteinase-13 in equine cartilage cocultured with synoviocytes

OBJECTIVE: To determine the effects of matrix metalloproteinase (MMP)-13, compared with interleukin (IL)-1alpha, on cartilage matrix molecule gene expression in a coculture system of equine cartilage explants and synoviocytes. SAMPLE POPULATION: Articular cartilage and synovium specimens harvested from femoropatellar joints of 4 horses, aged 3 to 5 years. PROCEDURES: Synoviocytes were isolated and cocultured with cartilage explants. Cultures were treated with human recombinant MMP-13 (1, 25, or 100 ng/mL) or IL-1alpha (0.01, 0.1, 1.0, or 10 ng/mL) for 96 hours, with medium exchange at 48 hours. Cartilage extracts and media were analyzed for glycosaminoglycan (GAG) content, and results were adjusted to cartilage DNA content. Quantitative PCR was performed on mRNA from cartilage (MMP-3, MMP-13, aggrecan, and collagen type IIB [COL2A1]) and synoviocytes (MMP-3 and MMP-13), and results were adjusted to 18S ribosomal subunit mRNA expression. Treatments were performed in triplicate, and the experiment was repeated 4 times. RESULTS: Cultures treated with MMP-13 or IL-1alpha had increased media GAG concentration at 48 and 96 hours. Aggrecan and COL2A1 mRNA expression were increased by application of MMP-13 or IL-1alpha. Gene expression of the catabolic mediator, MMP-3, in cartilage and synoviocytes was increased in cultures treated with MMP-13 or IL-1alpha. Expression of MMP-13 mRNA in cartilage was increased by IL-1alpha, but decreased in synoviocytes by MMP-13 treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the use of recombinant MMP-13 in a coculture system of synoviocytes and cartilage explants for the study of osteoarthritis.     

In vitro comparison of two techniques for suture prosthesis placement in the muscular process of the equine arytenoid cartilage

OBJECTIVE: To compare in vitro the load necessary for a partial and complete rupture of the muscular process arytenoid cartilage when a suture prosthesis is positioned by a bone trocar versus a trocar point needle and to compare failure mode. STUDY DESIGN: Experimental using cadaver specimens. SAMPLE POPULATION: Larynges from 18 Thoroughbred race horses, aged 2-20 years. METHODS: Arytenoid cartilages were separated randomly into 2 groups: group 1-suture prosthesis inserted directly through the muscular process using a curved trocar point needle and group 2-suture passed through a hole predrilled with a 3 mm bone trocar. Distracting force (constant rate, 1 mm/s) was applied to the suture until failure of the muscular process. Partial failure load, maximum load at complete failure, and force-time curve were recorded. Each arytenoid cartilage was examined, radiographed, and classified as having a linear or curved failure plane. RESULTS: No significant differences in mechanical test variables were detected. Failure mode followed the fissures occurring at the beginning of failure and then followed the tension axis. Significantly more linear failures occurred in group 2 (trocar) and more curved failures occurred in group 1 (needle). CONCLUSION: Use of a bone trocar for tunneling through the muscular process may reduce fissure formation. CLINICAL RELEVANCE: Use of bone trocar to create a hole in the muscular process of the arytenoid cartilage for suture passage in laryngoplasty may reduce fissure formation and decrease the risk of cartilage failure from suture pullout.     

Cellular heterogeneity in cathepsin D distribution in equine articular cartilage

The distribution of cathepsin D in normal equine growth cartilage has been examined immunocytochemically using an antiserum raised against human cathepsin D. The cross-reactivity and specificity of the antiserum for equine cathepsin D was confirmed, and its lysosomal localisation was demonstrated in horse skin fibroblasts by confocal scanning microscopy. Cultured horse chondrocytes were heterogenous in their expression of cathepsin D. Heterogeneity of distribution of the enzyme was also seen in chondrocytes in cartilage from different anatomical sites. A high level of cathepsin D was observed in the deep layer of cartilage from the lateral trochlear ridge of the distal femur. Cathepsin D was absent in the hypertrophic zone of the distal radial growth plate.     

Functional consequences of cartilage degeneration in the equine metacarpophalangeal joint: quantitative assessment of cartilage stiffness

REASONS FOR PERFORMING STUDY: No quantitative data currently exist on the relationship of the occurrence of cartilage degeneration and changes in site-specific biomechanical properties in the metacarpophalangeal (MCP) joint in the horse. OBJECTIVES: To gain insight into the biomechanical consequences of cartilage deterioration at 2 differently loaded sites on the proximal articular surface of the proximal phalanx (P1). HYPOTHESIS: Static and dynamic stiffness of articular cartilage decreases significantly in degenerated cartilage. METHODS: Cartilage degeneration index (CDI) values were measured at the lateral dorsal margin (Site 1), lateral central fovea (Site 2) and entire joint surface of P1 (CDIP1) in 30 horses. Group 1 contained joints without (CDIP1 values <25 %, n = 22) and Group 2 joints with (CDIP1 values >25 %, n = 8) signs of cartilage degeneration. Cartilage thickness at Sites 1 and 2 was measured using ultrasonic and needle-probe techniques. Osteochondral plugs were drilled out from Sites 1 and 2 and subsequently tested biomechanically in indentation geometry. Young's modulus at equilibrium and dynamic modulus were determined. RESULTS: Cartilage thickness values were not significantly different between the 2 groups and sites. Young's modulus at Site 1 was significantly higher in Group 1 than in Group 2; at Site 2, the difference was not significant. Dynamic modulus values were significantly higher in Group 1 than in Group 2 at both sites. CONCLUSIONS: Degenerative cartilage changes are clearly related to loss of stiffness of the tissue. Absolute changes in cartilage integrity in terms of CDI are greatest at the joint margin, but concomitant changes are also present at the centre, with a comparable decrease of the biomechanical moduli at the 2 sites. Therefore, significant cartilage degradation at the joint margin not only reflects local deterioration of biomechanical properties, but is also indicative of the functional quality in the centre. POTENTIAL RELEVANCE: These findings may be important for improving prognostication and developing preventative measures.     

Effects of dosage titration of methylprednisolone acetate and triamcinolone acetonide on interleukin-1-conditioned equine articular cartilage explants in vitro

REASONS FOR PERFORMING STUDY: Osteoarthritis is a frequent sequela of joint disease, especially with severe injuries or if attempts at therapy are unsuccessful. Negative and positive effects of corticosteroid treatment of articular cartilage have been demonstrated by in vitro and in vivo studies. OBJECTIVES: To assess the metabolic effects of varying dosages of methylprednisolone acetate (MPA) and triamcinolone acetonide (TA) on interleukin-1alpha (IL-1) conditioned equine cartilage explants. Our hypothesis was that lower dosages of corticosteroids would be less detrimental to cartilage metabolism than higher dosages. TA would be less detrimental to cartilage metabolism than MPA. METHODS: Treatment groups included articular cartilage explants with no IL-1 (control), IL-1 alone, and IL-1 plus 10, 5, 1 and 0.5 mg/ml MPA or 1.2, 0.6, 0.12 and 0.06 mg/ml TA. Explants were labelled with 35SO4 prior to the beginning and end of the experiment to assess glycosaminoglycan (GAG) degradation and synthesis, respectively. Total GAG content in media and explants and total cartilage DNA were also analysed. RESULTS: MPA and TA reduced GAG synthesis compared to control and IL-1 alone. The highest dosage of MPA (10 mg/ml) reduced GAG synthesis less than lower dosages of MPA and all dosages of TA. Compared to IL-1 alone, all dosages of TA and lower dosages of MPA increased GAG degradation. MPA at 10 mg/ml reduced GAG degradation. Both MPA and TA increased media GAG content compared to control and IL-1 explants. Total cartilage GAGs were unchanged with MPA, but reduced with TA, compared with IL-1 alone. Total cartilage DNA was decreased with MPA and increased with TA compared to IL-1 and control explants. CONCLUSIONS: MPA and TA did not counteract the negative effects of IL-1 and did not maintain cartilage metabolism at control levels. Lower dosages of MPA and TA were not less detrimental to cartilage metabolism than higher dosages. TA did not appear to be less harmful than MPA on cartilage metabolism. The results of this study differ from the findings of comparable in vivo studies. POTENTIAL RELEVANCE: The low numbers of horses used in this study limits extrapolation of these findings to the equine population; however, this study also questions the clinical relevance of this in vitro model.