The combination of mitogenic stimulation and DNA damage induces chondrocyte senescence.

OBJECTIVE: Cellular senescence is a phenotypic state characterized by stable cell-cycle arrest, enhanced lysosomal activity, and the secretion of inflammatory molecules and matrix degrading enzymes. Senescence has been implicated in osteoarthritis (OA) pathophysiology; however, the mechanisms that drive senescence induction in cartilage and other joint tissues are unknown. While numerous physiological signals are capable of initiating senescence, one emerging theme is that damaged cells convert to senescence in response to sustained mitogenic stimulation. The goal of this study was to develop an in vitro articular cartilage explant model to investigate the mechanisms of senescence induction. DESIGN: This study utilized healthy cartilage derived from cadaveric equine stifles and human ankles. Explants were irradiated to initiate DNA damage, and mitogenic stimulation was provided through serum-containing medium and treatment with transforming growth factor ß1 and basic fibroblastic growth factor. Readouts of senescence were a quantitative flow cytometry assay to detect senescence-associated ß galactosidase activity (SA-ß-gal), immunofluorescence for p16 and γH2AX, and qPCR for the expression of inflammatory genes. RESULTS: Human cartilage explants required both irradiation and mitogenic stimulation to induce senescence as compared to baseline control conditions (7.16% vs 2.34% SA-ß-gal high, p = 0.0007). These conditions also resulted in chondrocyte clusters within explants, a persistent DNA damage response, increased p16, and gene expression changes. CONCLUSIONS: Treatment of cartilage explants with mitogenic stimuli in the context of cellular damage reliably induces high levels of SA-ß-gal activity and other senescence markers, which provides a physiologically relevant model system to investigate the mechanisms of senescence induction.

Oral reserpine administration in horses results in low plasma concentrations that alter platelet biology.

BACKGROUND: Reserpine is a popular drug in the equine industry for long-term tranquilisation. Clinical observations revealed that blood from horses receiving oral reserpine was hypercoagulable. No studies have documented the pharmacokinetics of orally administered reserpine nor the effects of reserpine on platelets in horses. OBJECTIVES: To evaluate the pharmacokinetics of oral reserpine in horses and the effects of clinically relevant concentrations of reserpine on platelet functionality in vitro. STUDY DESIGN: Experimental controlled study. METHODS: The pharmacokinetics of oral reserpine (2.5 mg/horse, once) were determined in six healthy adult horses. Plasma samples were collected and concentrations of reserpine were determined by UPLC-MS/MS. Using this data, the in vitro effects of reserpine on platelets were examined. Aggregation, adhesion and releasate assays for serotonin and thromboxane B2 were performed on platelets exposed to varying concentrations of reserpine (0.01-10 ng/mL), aspirin (negative control) and saline (unexposed control). RESULTS: Oral reserpine administration demonstrated low plasma concentrations with a Cmax of 0.2 ± 0.06 ng/mL and a prolonged half-life of 23.6 ± 6.24 h. Simulations over a dose range of 2-8 µg/kg predicted Cmax at steady state between 0.06-0.9 ng/mL. Platelets exposed to these reserpine concentrations in vitro displayed increased aggregation and adhesion compared to unexposed or aspirin-exposed platelets as well as compared to higher concentrations of reserpine. These functional changes correlated with lower concentrations of serotonin and higher concentrations of thromboxane B2 in the platelet suspension supernatant. MAIN LIMITATIONS: This study used a small number of horses and only in vitro platelet experiments. CONCLUSIONS: Oral reserpine demonstrates low plasma concentrations and a prolonged half-life in horses. At these concentrations, reserpine causes significant changes in platelet function, most likely due to serotonin release and re-uptake which primes platelets for activation and thromboxane B2 release. These findings suggest that clinicians should harvest blood for biological processing prior to the onset of reserpine administration.

Allogeneic major histocompatibility complex-mismatched equine bone marrow-derived mesenchymal stem cells are targeted for death by cytotoxic anti-major histocompatibility complex antibodies.

BACKGROUND: Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Controversy exists, however, over whether major histocompatibility complex (MHC)-mismatched MSCs are recognised by the recipient immune system and targeted for death by a cytotoxic antibody response. OBJECTIVES: To determine if cytotoxic anti-MHC antibodies generated in vivo following MHC-mismatched MSC injections are capable of initiating complement-dependent cytotoxicity of MSCs. STUDY DESIGN: Experimental controlled study. METHODS: Antisera previously collected at Days 0, 7, 14 and 21 post-injection from 4 horses injected with donor MHC-mismatched equine leucocyte antigen (ELA)-A2 haplotype MSCs and one control horse injected with donor MHC-matched ELA-A2 MSCs were utilised in this study. Antisera were incubated with ELA-A2 MSCs before adding complement in microcytotoxicity assays and cell death was analysed via eosin dye exclusion. ELA-A2 peripheral blood leucocytes (PBLs) were used in the assays as a positive control. RESULTS: Antisera from all 4 horses injected with MHC-mismatched MSCs contained antibodies that caused the death of ELA-A2 haplotype MSCs in the microcytotoxicity assays. In 2 of the 4 horses, antibodies were present as early as Day 7 post-injection. MSC death was consistently equivalent to that of ELA-A2 haplotype PBL death at all time points and antisera dilutions. Antisera from the control horse that was injected with MHC-matched MSCs did not contain cytotoxic ELA-A2 antibodies at any of the time points examined. MAIN LIMITATIONS: This study examined MSC death in vitro only and utilized antisera from a small number of horses. CONCLUSIONS: The cytotoxic antibody response induced in recipient horses following injection with donor MHC-mismatched MSCs is capable of killing donor MSCs in vitro. These results suggest that the use of allogeneic MHC-mismatched MSCs must be cautioned against, not only for potential adverse events, but also for reduced therapeutic efficacy due to targeted MSC death.

Navigational ultrasound imaging: A novel imaging tool for aiding interventional therapies of equine musculoskeletal injuries.

REASONS FOR PERFORMING STUDY: Navigational ultrasound imaging, also known as fusion imaging, is a novel technology that allows real-time ultrasound imaging to be correlated with a previously acquired computed tomography (CT) or magnetic resonance imaging (MRI) study. It has been used in man to aid interventional therapies and has been shown to be valuable for sampling and assessing lesions diagnosed with MRI or CT that are equivocal on ultrasonography. To date, there are no reports of the use of this modality in veterinary medicine. OBJECTIVES: To assess whether navigational ultrasound imaging can be used to assist commonly performed interventional therapies for the treatment of equine musculoskeletal injuries diagnosed with MRI and determine the appropriateness of regional anatomical landmarks as registration sites. STUDY DESIGN: Retrospective, descriptive clinical study. METHODS: Horses with musculoskeletal injuries of the distal limb diagnosed with MRI scheduled for ultrasound-guided interventional therapies were evaluated (n = 17 horses with a total of 29 lesions). Anatomical landmarks used for image registration for the navigational procedure were documented. Accuracy of lesion location and success of the procedure were assessed subjectively and described using a grading scale. RESULTS: All procedures were accurately registered using regional anatomical landmarks and considered successful based on our criteria. Anatomical landmarks were described for each lesion type. The addition of navigational imaging was considered to greatly aid the procedures in 59% of cases and added information to the remainder of the procedures. The technique was considered to improve the precision of these interventional procedures. CONCLUSIONS: Navigational ultrasound imaging is a complementary imaging modality that can be used for the treatment of equine soft tissue musculoskeletal injuries diagnosed with MRI.

Plasma and synovial fluid concentration of doxycycline following low-dose, low-frequency administration, and resultant inhibition of matrix metalloproteinase-13 from interleukin-stimulated equine synoviocytes.

REASONS FOR STUDY: To determine whether low-dose, low-frequency doxycycline administration is capable of achieving chondroprotective concentrations within synovial fluid (SF) while remaining below minimum inhibitory concentration 90 (MIC90 ) of most equine pathogens and would be an option in the management of osteoarthritis. OBJECTIVES: To determine whether low-dose, low-frequency oral administration of doxycycline can attain in vivo SF concentrations capable of chondroprotective effects through reduction of matrix metalloproteinase (MMP)-13 activity, while remaining below MIC90 of most equine pathogens. STUDY DESIGN: Descriptive pharmacokinetic study with crossover design. METHODS: Two groups of 6 horses received oral doxycycline. Plasma and SF doxycycline concentrations were measured using high performance liquid chromatography. Group 1 received 5 mg/kg bwt q. 24 h with 21 blood and 8 SF samples collected over 120 h; Group 2 received 5 mg/kg bwt q. 48 h with 27 blood and 11 SF samples collected over 192 h. Cultured synoviocytes were treated with interleukin-1α (1 ng/ml) for 24 h to stimulate MMP synthesis, and then SF was added to the culture medium for 96 h. MMP-13 protein and mRNA were measured in synoviocyte culture medium and synoviocytes, respectively. RESULTS: Mean doxycycline concentration ≥0.043 µg/ml (previously demonstrated to inhibit MMP-13) was achieved in plasma by t = 0.25 h and SF by t = 48 h in Group 1, and in plasma by t = 0.17 h and SF by t = 1 h in Group 2. Synoviocyte culture medium containing doxycycline from Groups 1 and 2 had significantly decreased active MMP-13 protein concentration, and synoviocytes cultured in this medium had significantly decreased MMP-13 gene expression compared to controls. Plasma doxycycline concentration in both groups and SF doxycycline concentration in Group 2 demonstrated a cumulative effect. CONCLUSIONS: Low-dose orally administered doxycycline achieves SF concentrations in vivo capable of diminishing MMP-13 expression. This study supports the use of doxycycline as a disease modifying osteoarthritic drug.

Pharmacokinetics and distribution of minocycline in mature horses after oral administration of multiple doses and comparison with minimum inhibitory concentrations.

REASONS FOR PERFORMING STUDY: Minocycline holds great potential for use in horses not only for its antimicrobial effects but also for its anti-inflammatory and neuroprotective properties. However, there are no pharmacokinetic or safety data available regarding the use of oral minocycline in horses. OBJECTIVES: To determine pharmacokinetics, safety and penetration into plasma, synovial fluid, aqueous humour (AH) and cerebral spinal fluid (CSF) of minocycline after oral administration of multiple doses in horses and to determine the minimum inhibitory concentrations (MIC) of minocycline for equine pathogenic bacteria. METHODS: Six horses received minocycline (4 mg/kg bwt q. 12 h for 5 doses). Thirty-three blood and 9 synovial fluid samples were collected over 96 h. Aqueous humour and CSF samples were collected 1 h after the final dose. Minocycline concentrations were measured using high pressure liquid chromatography. The MIC values of minocycline for equine bacterial isolates were determined. RESULTS: At steady state, the mean ± s.d. peak concentration of minocycline in the plasma was 0.67 ± 0.26 µg/ml and the mean half-life was 11.48 ± 3.23 h. The highest trough synovial fluid minocycline concentration was 0.33 ± 0.12 µg/ml. The AH concentration of minocycline was 0.09 ± 0.03 µg/ml in normal eyes and 0.11 ± 0.04 µg/ml in blood aqueous barrier-disrupted eyes. The mean CSF concentration of minocycline was 0.38 ± 0.09 µg/ml. The MIC values were determined for 301 isolates. Minocycline concentrations were above the MIC(50) and MIC(90) for many gram-positive equine pathogens. POTENTIAL RELEVANCE: This study supports the use of orally administered minocycline at a dose of 4 mg/kg bwt every 12 h for the treatment of nonocular infections caused by susceptible (MIC ≤ 0.25 µg/ml) organisms in horses. Further studies are required to determine the dose that would be effective for the treatment of ocular infections.

Size and geometry of apical sesamoid fracture fragments as a determinant of prognosis in Thoroughbred racehorses.

REASON FOR PERFORMING STUDY: Analysis was performed to examine a method for refining the preoperative prognosis for horses that had surgery to remove apical fractures of the proximal sesamoid bones (PSBs). OBJECTIVES: To determine if: 1) there was a difference in size or configuration of apical fractures between the different anatomical locations of the PSBs, which have been shown to affect the prognosis; and 2) the size or configuration could predict the prognosis for racehorses with these fractures. METHODS: The study included 110 weanlings and yearlings and 56 training racehorses that underwent surgery to remove apical PSB fractures. Radiographs of the fractures were used for measurement of the abaxial and axial proportion and the abaxial to axial ratio, and race records were used to determine average earnings per start (AEPS) and total post operative starts. Analysis of variance and regression statistics were used to compare the fragment sizes between the specific PSBs on each of the limbs and compare size and configuration of the fractures to prognosis. RESULTS: There was a significantly larger abaxial to axial ratio (more transverse fracture) for the forelimb medial sesamoids than for all other sesamoids in untrained racehorses (P = 0.03). There were no other significant differences in size. There was no relationship between fracture size or configuration and AEPS nor total post operative starts. CONCLUSIONS: Apical fractures in weanlings and yearlings tend to be more transverse in the forelimb medial PSBs than the other PSBs. Apical fracture size and geometry does not determine prognosis for apical sesamoid fractures. POTENTIAL RELEVANCE: Horses that undergo surgery to remove larger apical fractures of the PSBs do not have a worse outcome than those horses with smaller fractures.

Orally administered doxycycline accumulates in synovial fluid compared to plasma.

REASONS FOR PERFORMING STUDY: Tetracycline compounds have been used to slow the progression of osteoarthritis (OA) and rheumatoid arthritis but the concentration of doxycycline attained in synovial fluid following oral, low-dose administration has yet to be determined. OBJECTIVE: To determine the concentration of doxycycline in synovial fluid following oral, low-dose administration. METHODS: Six mature horses received doxycycline (5 mg/kg bwt q. 12 h for 5 doses). Venous blood and synovial fluid samples were collected at t=0, 0.25, 0.5, 1, 12, 24, 48 and 72 h. Doxycycline concentrations were measured using reverse phase high pressure liquid chromatography with ultraviolet detection. RESULTS: Doxycycline concentrations at all time points after t=0 were above the lower limit of quantification for the assay. Plasma concentrations of doxycycline were above 0.21 microg/ml at t=0.5 h. The mean+/-s.d. peak concentration (Cmax) of doxycycline in plasma was 0.37+/-0.22 microg/ml and time to peak concentration was 0.54+/-0.19 h. Synovial fluid concentrations of doxycycline were above 0.12 microg/ml 1 h after drug administration. The mean Cmax of doxycycline in the synovial fluid was 0.27+/-0.10 microg/ml. The penetration factor of doxycycline from plasma into synovial fluid, as determined by a ratio of the area-under-the-curve for synovial fluid:plasma during the sampling period, was 4.6. POTENTIAL RELEVANCE: Orally administered doxycycline distributes easily into synovial fluid with a penetration factor of 4.6. Terminal half-life of the drug in synovial fluid was longer than in the plasma, indicating possible accumulation in this compartment. Further in vivo studies are warranted to define a medication protocol prior to routine clinical use of doxycycline for the treatment of OA.

Effects of platelet rich plasma and acellular bone marrow on gene expression patterns and DNA content of equine suspensory ligament explant cultures.

REASONS FOR PERFORMING STUDY: Suspensory ligament (SL) desmitis is a common source of lameness. The results of this study will determine if blood-derived products stimulate SL matrix synthesis and have potential as regenerative therapies for SL desmitis OBJECTIVES: To determine if various blood-based biological products including plasma, blood, PRP, platelet poor plasma (PPP) and ABM aspirate stimulates anabolic and/or catabolic pathways in suspensory ligaments (SL). METHODS: The body of the SL was harvested from 6 horses and used to establish explant cultures. Explants were cultured in plasma, blood, PRP, PPP or ABM at concentrations of 10, 50 or 100%. Anabolic responses were assessed by use of quantitative PCR for collagens type I and III, cartilage oligomeric matrix protein (COMP) and decorin. Total DNA and collagen protein content were also measured. Catabolic reactions were measured by quantitative PCR for matrix metalloproteinases 3 and 13 (MMP-3, MMP-13). RESULTS: Acellular bone marrow aspirate at 100% stimulated decorin and COMP mRNA synthesis more than all other treatments at all concentrations. No treatment at any concentration stimulated the catabolic gene MMP-13; only 50% ABM stimulated MMP-13 mRNA expression. CONCLUSIONS: Acellular bone marrow is indicated, and might be preferred to plasma, blood or PPP, as a blood-based biological source for SL tissue regenerative therapy. Long-term, placebo controlled case studies are indicated to determine if ABM aids in recovery from SL desmitis. POTENTIAL RELEVANCE: Bone marrow aspirate is an autogenous, readily available biological source for SL regenerative therapy where the aim is to stimulate matrix synthesis.

Racing performance after arthroscopic removal of apical sesamoid fracture fragments in Thoroughbred horses age < 2 years: 151 cases (1989--2002).

REASONS FOR PERFORMING STUDY: Studies on arthroscopic removal of apical proximal sesamoid fracture fragments in Thoroughbred (TB) horses age > or = 2 years have reported a high success rate. However, there are no reports documenting the racing prognosis of TB horses that undergo such surgery as weanlings or yearlings. OBJECTIVES: To describe the incidence of apical proximal sesamoid fractures in immature TB horses, age < 2 years and determine probability and quality of racing performance after arthroscopic removal of such fractures in 151 TB weanlings and yearlings. METHODS: The medical records of TB horses age < 2 years that underwent arthroscopic surgery for removal of apical proximal sesamoid fracture fragments were reviewed. Follow-up information was obtained from race records. Student's t tests were used to compare performance variables of operated racehorses to that of their maternal siblings. RESULTS: Ninety-two percent (139/151) of fractures occurred in the hindlimbs and 8% (11/151) in the forelimbs (fracture of both fore- and hindlimb, n = 1). Horses with forelimb fractures had a greatly reduced probability of racing (55%) compared to those with hindlimb fractures (86%). Overall, 84% of the horses raced post operatively and had performance records similar to that of their maternal siblings, 78% (787/1006) of which raced. CONCLUSIONS: Arthroscopic removal of apical proximal sesamoid fracture fragments in TB weanlings and yearlings carries an excellent prognosis for racing in horses with hindlimb fractures and a reduced prognosis in those with forelimb fractures. Medial fractures of the forelimb carry the worst prognosis. POTENTIAL RELEVANCE: The determination of prognosis increases knowledge on apical sesamoid bone fractures and potential for arthroscopic restoration of the ability to race; and enables the value of yearlings for subsequent sale to be established.

Racing performance after arthroscopic removal of apical sesamoid fracture fragments in Thoroughbred horses age > or = 2 years: 84 cases (1989-2002).

REASONS FOR PERFORMING STUDY: Studies have shown that surgical removal of apical fracture fragments in Standardbred racehorses carries the best prognosis for return to racing performance, but there are no reports involving mature Thoroughbred (TB) racehorses. OBJECTIVES: To describe the incidence of apical proximal sesamoid fractures in TB racehorses and determine probability and quality of racing performance after arthroscopic removal of such fractures in TB racehorses age > or = 2 years. METHODS: Medical records and pre- and post operative race records of TB racehorses age > or = 2 years that underwent arthroscopic surgery for removal of apical proximal sesamoid fracture fragments were reviewed. RESULTS: Sixty-four percent of fractures occurred in the hindlimbs and 36% in the forelimbs. Horses with forelimb fractures had a reduced probability of return to racing (67%) compared to those with hindlimb fractures (83%), but the majority (77%) of treated horses recovered to return to race post operatively. Horses with medial forelimb fractures raced at only a 47% rate; those with suspensory desmitis at 63%. Unlike Standardbreds, there was no difference in probability of racing post operatively between horses that had, and had not, raced preoperatively. CONCLUSIONS: Data show that arthroscopic removal of apical proximal sesamoid fracture fragments is successful at restoring ability to race in skeletally mature TB horses without evidence of severe suspensory ligament damage. Prognosis for return to racing is excellent (83%) in horses with hindlimb fractures and good (67%) in those with forelimb fractures. Medial fractures of the forelimb have the worst prognosis. POTENTIAL RELEVANCE: The determination of prognosis for differing sites in TB racehorses should increase knowledge of apical proximal sesamoid bone fractures and improve communication from veterinarian to owner, and trainer, on the potential for arthroscopic restoration of the ability to race.